Etiopatogénese da contratura
capsular de próteses mamárias
Etiopathogenesis of capsular contracture in breast implants
Carmen Marisa Marques Gonçalves
Assistente Convidada da Faculdade de Medicina da Universidade do Porto
Faculdade de Medicina, Universidade do Porto
Assistente Hospitalar de Cirurgia Plástica, Reconstrutiva e Estética
Serviço de Cirurgia Plástica, Reconstrutiva, Estética e Maxilo-Facial/Unidade de Queimados
Centro Hospitalar de São João, Porto
ORIENTADOR
Professor Doutor José Manuel Lopes Teixeira Amarante
Professor Catedrático da Faculdade de Medicina da Universidade do Porto
Faculdade de Medicina, Universidade do Porto
Serviço de Cirurgia Plástica, Reconstrutiva, Estética e Maxilo-Facial /Unidade de Queimados
Centro Hospitalar de São João, Porto
CO-ORIENTADOR
Professor Doutor Acácio Agostinho Gonçalves Rodrigues
Professor Associado da Faculdade de Medicina da Universidade do Porto
Serviço de Microbiologia
Faculdade de Medicina, Universidade do Porto
Serviço de Cirurgia Plástica, Reconstrutiva, Estética e Maxilo-Facial /Unidade de Queimados
Centro Hospitalar de São João, Porto
Dissertação de candidatura ao grau de Doutor apresentado à Faculdade de Medicina da
Universidade do Porto
Academic dissertation, to be presented, with the permission of the Faculty of Medicine
of the University of Porto, for public examination
Porto, 2012
Orientador
Doutor José Manuel Lopes Teixeira Amarante
Professor Catedrático da Faculdade de Medicina da Universidade do Porto
Co-orientador
Doutor Acácio Agostinho Gonçalves Rodrigues
Professor Associado da Faculdade de Medicina da Universidade do Porto
Júri
Presidente - Doutor José Agostinho Marques Lopes
Diretor da Faculdade de Medicina da Universidade do Porto
Professor Catedrático da Faculdade de Medicina da Universidade do Porto
Vogais - Doutor Spencer Austin Brown
Professor da University of Pittsburg, Pennsylvania
Doutor Manuel do Rosário Caneira da Silva
Professor Auxiliar Convidado da Faculdade de Medicina da Universidade
de Lisboa
Doutor José Rosa de Almeida
Professor Auxiliar Convidado da Faculdade de Ciências Médicas da
Universidade Nova de Lisboa
Doutora Maria Amélia Duarte Ferreira
Professora Catedrática da Faculdade de Medicina da Universidade do Porto
Doutor José Manuel Lopes Teixeira Amarante
Professor Catedrático da Faculdade de Medicina da Universidade do Porto
1
2
Supervised by
Professor José Manuel Lopes Teixeira Amarante, MD, PhD
Department of Plastic Surgery
Faculty of Medicine, University of Oporto
Centro Hospitalar of São João
Oporto, Portugal
Professor Acácio Agostinho Gonçalves Rodrigues, MD, PhD
Department of Microbiology
Faculty of Medicine, University of Oporto
Department of Plastic Surgery
Centro Hospitalar of São João
Oporto, Portugal
Jury
President - José Agostinho Marques Lopes, MD, PhD
Vowels - Spencer Austin Brown, BA, PhD
Manuel do Rosário Caneira da Silva, MD, PhD
José Rosa de Almeida, MD, PhD
Maria Amélia Duarte Ferreira, MD, PhD
José Manuel Lopes Teixeira Amarante, MD, PhD
3
4
Contents
List of original publications
7
1.Introduction
11
2. Aims of the thesis
3. Material and methods
43
45
4. Results
59
5. Discussion
95
Abbreviations
9
1.1Etiology of capsular contracture
1.2 Classification of capsular contracture
1.2.1 Baker classification rates and follow-up
1.3 Estrogens
1.3.1 Estrogens review of literature
1.4 The subclinical infection in the development of capsular contracture
1.5 Histology and capsular pressure
1.6 The immunology of fibrosis
1.6.1 Pathophysiological hallmarks of breast implant capsule formation
1.6.2 Microdialysis , IL-8 and TNF-alpha
1.7 Prevention and treatment of capsular contracture
1.7.1 Tissucol/Tisseel®
1.7.2 FloSeal®
1.7.3 Triamcinolone acetonide
1.8. Chitosan and Chitooligosaccharides
1.9 The New Zealand white rabbit
1.10 The pig and the mice models
STUDY 1
STUDY 2
STUDY 3
STUDY 4
STUDY 5
14
15
17
19
19
21
22
25
27
30
31
33
35
36
37
39
41
45
47
51
53
56
STUDY 1
STUDY 2
STUDY 3
STUDY 4
STUDY 5
59
70
78
85
90
STUDY 1
STUDY 2
STUDY 3
STUDY 4
STUDY 5
95
97
101
106
114
6. Conclusions
121
Original publications
151
Financial disclosure and products page
Acknowledgements
References
123
125
131
5
6
List of original publications
This thesis is based on the following publications which are referred in the text by their
Roman numerals I-VI:
I- Adams, WP., Haydon, MS., Raniere, J., Trott S., Marques, M., Feliciano, M.,
Robinson, JB., Tang, L., Brown, SA. A rabbit model for capsular contracture:
development and clinical implications. Plast Reconstr Surg. 2006; 117: 1214-9.
Plastic and Reconstructive Surgery journal is indexed by Thomson Reuters (ISI); the journal's
impact factor is 2,647 the highest in Plastic Surgery. The publication`s Scopus times cited is 15.
The publication was presented in the XXXIV Reunião da Sociedade Portuguesa de Cirurgia
Plástica, Reconstrutiva e Estética, Oporto, Portugal, 2004.
II- Marques, M., Brown, SA., Oliveira, I., Cordeiro, N., Morales-Helgera, A.,
Gonçalves-Rodrigues, A., Amarante, J. Long-term follow-up of breast capsule
contracture rates in cosmetic and reconstructive cases. Plast Reconstr Surg. 2010; 126:
769-778.
Plastic and Reconstructive Surgery journal is indexed by Thomson Reuters (ISI); the journal's
impact factor is 2,647 the highest in Plastic Surgery. The publication`s Scopus times cited is 4
and was the most popular publication in this journal in September 16, 2010.
The publication was presented in the XL Reunião Anual da Sociedade Portuguesa de Cirurgia
Plástica Reconstrutiva e Estética, Coimbra, Portugal, 2010.
III-Marques, M., Brown, SA., Cordeiro, N., Rodrigues-Pereira, P., Cobrado, L.,
Morales-Helgera, A., Lima, N., Luís, A., Mendanha, M., Gonçalves-Rodrigues, A.,
Amarante, J. Effects of fibrin, thrombin and blood on breast capsule formation in a preclinical model. Aesthet Surg J. 2011; 31: 302-309.
Aesthet Surgery Journal is indexed with Thomson Reuters (ISI). The publication`s Scopus times
cited is 2.
The publication was presented in the XL Reunião Anual da Sociedade Portuguesa de Cirurgia
Plástica Reconstrutiva e Estética, Coimbra, Portugal, 2010 and received the prize of the “Best
Aesthetic Surgery Communication” to represent Portugal in the “Voice of Europe”, 4º European
Association of the Societies of Aesthetic Plastic Surgery (EASAPS) Congress.
IV-Marques, M., Brown, SA., Cordeiro, N., Rodrigues-Pereira, P., Cobrado, L.,
Morales-Helgera, A., Queirós, L., Luís, A., Freitas, R., Gonçalves-Rodrigues, A.,
Amarante, J. Effects of coagulase negative staphylococci and fibrin on breast capsule
formation in a rabbit model. Aesthet Surg J. 2011; 3: 420-428.
Aesthet Surgery Journal is indexed by Thomson Reuters (ISI). The publication`s Scopus times
cited is 2.
The publication was presented in the XL Reunião Anual da Sociedade Portuguesa de Cirurgia
Plástica Reconstrutiva e Estética, Coimbra, Portugal, 2010, and received the prize of the “Best
Aesthetic Surgery Communication” simultaneously with communication above.
Both publications (III and IV) represented Portugal in the “Voice of Europe”, EASAPS
Congress, Milan, Italy, 2011.
7
V-Marques, M., Brown, SA., Rodrigues-Pereira, P., Cordeiro, N., Morales-Helgera, A.,
Cobrado, L., Queirós, L., Freitas, R., Fernandes, J., Correia-Sá, I., GonçalvesRodrigues, A., Amarante, J. Animal Model of Implant Capsule Contracture: effects of
chitosan. Aesthet Surg J. 2011; 31: 540-550.
Aesthet Surgery Journal is indexed by Thomson Reuters (ISI). The publication`s Scopus times
cited is 0.
The publication was presented in the XL Reunião Anual da Sociedade Portuguesa de Cirurgia
Plástica Reconstrutiva e Estética, Coimbra, Portugal, 2011.
VI- Marques, M., Brown, SA., Cordeiro, N.D.S., Rodrigues-Pereira, P., GonçalvesRodrigues, A., Amarante, J. The impact of triamcinolone-acetonide in early breast capsule
formation, in a rabbit model. Aesthetic Plastic Surgery. 2012; Apr.
Aesthetic Plastic Surgery journal is indexed by Thomson Reuters (ISI); the journal's impact
factor is 1,252. The publication`s Scopus times cited is 0.
The publication was presented in the I Congresso Ibero-Escandinavo de Cirurgia Plástica
Reconstrutiva e Estética / XLVII Congresso Nacional da Sociedade Espanhola de CPRE, Palma
de Mallorca, Spain, 2012.
Abstract publication
Marques, M. Effects of fibrin (Tisseel/Tissucol®) on breast capsule formation in a rabbit
model. Aesthetic Plastic Surgery. 2012; Jun.
The publication was presented in the Voice of Europe 2011 as the Voice of Portugal (from
EASAPS Milan Congress).
Aesthetic Plastic Surgery journal is a publication of the International Society of Aesthetic Plastic
Surgery and the official journal of the European Association of Societies of Aesthetic Plastic
Surgery (EASAPS). Aesthetic Plastic Surgery journal is indexed by Thomson Reuters (ISI); the
journal's impact factor is 1,252.
8
Abbreviations
CD: cluster of differentiation
CHAID: Chisquared Automatic Interaction Detection
CI: confidence intervals
COS: chitooligosaccharide
CS: chitosan
CTGF: connective tissue growth factor
DCs: dendritic cells
ECM: collagenous extracellular matrix
EGF: epidermal growth factor
ET-1: endothelin 1
bFGF: basic fibroblast growth factor
HSP 60: heat shock proteins 60
IC: inhibitory concentration
ICAM-1: intercellular adhesion molecule 1
IL: interleukin
IFN-y: interferon y
IGF-1: insulin like growth factor-1
LMWC: low molecular weight chitosan
LPS: lipopolysaccharide
MCP-1 : monocyte chemotactic protein-1
f-MLP: formyl-methionyl-leucyl-phenylalanine
MALP-2: macrophage activating lipopeptide 2
MMP: macrophage-derivated matrix metalloproteinases
MPI-1α : macrophage inflammatory proteins 1α
NK: Natural killer
NO: oxide
iNOS: nitric oxide synthase
OPN: osteopontin
OSM: oncostatin M
PDGF: platelet-derived growth factor
PEGA: polyethylene glycol adipate
PF-4: plated factor 4
PMN: polymorphonuclear leukocytes
RANTES: regulated upon activation normal T-cell expressed presumed secreted
ROS: reactive oxygen species
RR: relative risks
α-SMA: α-smooth muscle cell actine
SPSS: Statistical Package for Social Sciences
9
TA: triamcinolone acetonide
TDA: toluenediamine
TDI: toluene diisocyanate
TGF-β1: transforming growth factor beta 1
Th: distinct types of T-helper cells
TNF-α: Tumor necrosis factor alpha
VCAM-1: vascular cell adhesion molecule 1
VEGF: vascular endothelial growth factor
WHI: Women´s Health Initiative
10
1. Introduction
Silicone gel breast implants have been implanted world-wide, for cosmetic
augmentation and breast reconstruction, since 1962[1]. Research studies have focused
on the potential adverse health effects of silicone implants, particularly, possible links
with cancer or connective tissue disorders, but none have yet shown an increased risk of
other diseases associated with those implants[2],[3],[4],[5],[6],[7],[8],[9],[10],[11]. Additional
reports have focused on postoperative local complications, and patient safety issues in
women receiving silicone breast implants[12],[13],[14],[15],[16],[17],[18],[19].
Capsular contracture is the formation of fibrous scar tissue investing a foreign
body or surgically implanted device and is the most common severe chronic
complication associated with silicone breast implants[12],[13],[14],[15],[16],[17],[18],[19], with a
clinical realistic incidence ranging from 8 to 45%[20],[21],[22],[23],[24],[25].
Silicone breast implants have been certified according to European Union’s
safety and efficacy requirements as class III medical devices and reclassified by the
European Union into the strictest category of medical devices for sale to the public.
Silicone breast implants are the most widely applied medical implants in European
countries. Saline-based implants, on the other hand, have been almost exclusively used
in North America and are related to much more complications than silicone-based
implants, such us rippling, firmer consistency, leaking and complete deflation[26]. So far,
there is a lack of current prospective data comparing capsule contracture with saline
versus silicone breast implants[27]. Recently McCarthy et al. [28] concluded in the setting
of postmastectomy reconstruction, patients who received silicone breast implants (n =
176) reported significantly higher satisfaction with the results of reconstruction than
those who received saline implants (n = 306). The first-generation of silicone implants
had a thick shell and viscous gel, while the second-generation possessed a much thinner
11
gel and shell[29]. However, with these implants, excessive silicone gel bleed and rupture
rates occurred[30]. The development of the third-generation gave birth to the “low bleed”
implant containing a barrier-coated shell[29]. Ever since polyurethane-coated implants
were
reported
to
be
associated
with
lower
rates
of
capsular
contracture[31],[32],[33],[34],[35],[36], the use of breast implants with a textured surface have
been the subject of recent reviews[37].
Several studies have shown that textured implants have a lower tendency to
develop capsular contracture than smooth-surface implants[22],[38],[39],[40],[41],[42], although
others have reported the opposite[43],[44],[45]. In addition, there is substantial evidence that
placement in the subglandular plane is associated with a higher incidence of capsular
contracture[46] and is less satisfactory for mammography[47]. On the contrary, other
studies show a lower proportion of capsular contracture, which was not however
statistically significant[22],[48]. That observation may be attributed to a greater difficulty
in appreciating capsular contracture in a deeper submuscular plane or to the fact that
textured surface implants have no impact on capsular contracture when placed
submuscularly.
The polyurethane foam-covered breast implant, a silicone gel-filled device
surrounded by a 1- to 2mm-thick layer of polyurethane foam, is associated with a lower
incidence of capsular contracture[49],[50]. In the late 1980s it was reported that in vitro
degradation of polyurethane could lead to formation of substances known to be
carcinogenic in animals[51]. In a retrospective study comprised of individuals receiving
either polyurethane breast implants (n = 568) or other types of silicone gel-filled breast
implants (n = 963), between 1981 and 2004 (23 years), Handel[52] concluded that the
incidence of capsular contracture was dramatically lower with polyurethane foamcovered implants compared to smooth or textured implants. This beneficial effect
12
persisted at least 10 years after implantation. Aside from skin rash, the polyurethane
foam-covered implants appear to have a safety profile similar to other silicone gel-filled
devices. The polyurethane used in the manufacture of breast implants is a
polyesterurethane made from polyethylene glycol adipate (PEGA) and toluene
diisocyanate (TDI). The TDI is unstable in an aqueous environment and converts slowly
into toluenediamine (TDA). The carcinogenic effect of toluenediamine (TDA) has never
been established in humans[53]. To quantify in vivo release of TDA, Hester et al.[54]
collected urine and serum samples from 61 patients with polyurethane foam-covered
implants and 61 controls on two occasions separated by 10 +/- 3 days. No patients or
controls had detectable free 2,4-TDA in their sera. Eighteen patients with polyurethane
foam-covered implants had detectable levels in their urine, compared to 7 control
subjects. The biodegradative half-life of the polyurethane foam was estimated to be 2
years. The risk assessment of approximately one in one million derived from this study
strengthens earlier conclusions by the Health Protection Branch (Canada) that there is
no significant risk of cancer from exposure to the 2,4-TDA formed from this
biodegradation. In a review of the literature, McGrath and Burkhardt[55] concluded there
was no evidence to link breast implants of any kind with an increased risk of breast
cancer. So far, the polyurethane foam-covered breast implants are not FDA-approved in
the USA. One reason is the fact that the polyurethane disappears over time; the lack of a
surgical plane of dissection, high rate of intraoperative bleeding, and difficult
postexplantation reconstruction make this operation very demanding[56]. Where
polyurethane goes and what effects it might cause will take many years of study to
answer. The fact that we are again noticing an interest in using polyurethane implants
should remind us of the real problem of capsule contracture with silicone gel breast
implants.
13
In a consecutive, population-based study consisting of 1529 patients receiving
3495 implants at a multidisciplinary breast center between 1979 and 2004 (25 years), by
Handel et al.[57], the authors concluded: 1) the longer implants were in place, the greater
the cumulative risk of developing contracture, consistent with other studies[9],[20],[58],[59];
2) hematoma significantly increased the risk of contracture, consistent with other
studies[16],[60]; 3) smooth and textured implants had similar contracture rates,
controversial in many studies[22],[38],[39],[40],[41],[42],[43],[44],[45]; 4) polyurethane foamcovered implants had a reduced risk of contracture persisting for at least 10 years after
implantation, consistent with other studies[31],[32],[33],[34],[35],[36],[52]. On a systemic review
of the literature by Shaub, Ahmad and Rohrich[27], the authors were unable to conclude
which implants, silicone versus saline, have a higher incidence of CC.
1.1 Etiology of capsular contracture
The true etiology and subsequent treatment of capsular contracture remains yet
elusive.
Two
prevailing
theories
emerged[21],[39],[61],[62],[63],[64],[65],[66],[67],[68],[69],[70],[71],[72],[73],[74],[75],[76]:
have
the
infectious
hypothesis and the hypertrophic scar hypothesis. The infectious hypothesis, which has
been championed by Burkhardt[61],[63] , supported by others[71],[72],[73],[74],[76],[77],[78],[79] and
more recently studied by Rohrich and Adams et al
[21],[62],[64]
, implicated subclinical
infection in the development of capsular contracture. The hypertrophic scar
hypothesis[61],[68],[69],[70],[75],[76],[80],[81],[82],[83], implicated non-infectious stimuli, namely
hematoma, granuloma, or hereditary factors, which may confer a foreign body reaction
and result in formation of a hypertrophic scar around an implanted device.
In our opinion the cause of capsular contracture is multifactorial. We purpose
this point of view in the publication I[84], which was refuted by Burkhardt in the
14
discussion of this paper. The purpose of this thesis is to clarify its etiology as an
extension of this first publication.
1.2 Classification of capsular contracture
The tables I[25] and II[78] describe the two classifications of capsular contracture.
Because the Baker classification is widely used, it will be the one reported in this thesis.
Table I. Baker Classification
Grade
Description
I
Breast absolutely natural, no one could tell breast was augmented
II
Minimal contracture; surgeon can tell surgery was performed but patient
has no complaint
III
Moderate contracture; patient feels some firmness
IV
Severe contracture; obvious just from observation
Table II. Breast Augmentation Classification
Grade
Description
I
Soft; no deformation
II
Slightly thickened consistency; none to slight deformation
III
Firm to hard; none to slight deformation
IV
Hard; severe deformation
15
Con de
nt
i
a
l
Con de
nt
i
a
l
Con de
nt
i
a
l
The Baker classification system defines stages of breast capsule clinical
presentation into distinct grades[25]. Grade II (Figure 1 a/b/c) is the first stage of capsular
contracture and clinical interpretation of grade II may be highly dependent on individual
surgeons’ opinions. Although the clinical impact of grade II is relevant to the continuum
of breast capsule formation, the majority of retrospective and prospective reports do not
include grade II subjects as breast capsule cases[85],[86],[87],[88]. The exclusion of grade II
subjects in these reports may result in under-reporting of capsular contracture rates. The
purpose of this thesis (Study 1) was to report the incidence of complications with breast
implants in a Portuguese population, in aesthetic and reconstructive groups and to
perform a comprehensive evaluation of the importance of grade II and the follow-up
time period. In this study we analyse the possible associations among surgical route,
implant placement, body index mass, smoking habits, alcohol consumption, and
capsular contracture.
1.2.1 Baker classification rates and follow-up
Table III[12],[13],[14],[18],[19],[57]
,[85],[86],[87],[88],[89],[90],[91],[92],[93],[94]
demonstrates that
reported capsular contracture rates vary widely due to authors’ reporting Baker
classification and follow-up time periods. These data showed incidence complications
were elevated in reconstruction patients compared to cosmetic augmentation
patients[13],[95].
17
Table III. Studies average follow-up versus capsular contracture
Studies
Type of
study
Prospective
Number of patients
Adams et al.,
2006
Prospective
235 (172 cosmetic
primary augmentation;
63 reconstructive)
14 months
Henriksen et al.,
2005
Brown et al.,
2005
Retrospective
2277
19.5 months
Retrospective
150 (118 cosmetic; 32
reconstructive)
21 months
Fruhstorfer et al.,
2004
Henriksen et al.,
2003
Cunningham et
al., 2007
Prospective
35
23 months
Cosm: 2 cases
Reconst: 3 cases
Just Baker II; no cases Baker IIIIV
0%
Prospective
1090
2 years
4.1% (Baker II-IV)
Prospective
955 (572 primary
augmentation ; 123
revisionsaugmentation ; 191
reconstruction ; 69
revisionsreconstruction)
830
2 years
Baker III-IV:
0.8% primary augmentation
5.4 revisions-augmentation
2.39 years
0%
44
1007 (551 primary
augmentation ; 146
revisionsaugmentation ; 251
reconstruction ; 59
revisionsreconstruction)
941 (492 cosmetic
primary augmentation;
225 reconstructive;
224 revisions)
940 (455 cosmetic
primary augmentation;
98 reconstructive; 162
revisions)
34 months
3 years
20% (Baker II-IV)
Baker III-IV:
8.1% primary augmentation
18.9 revisions-augmentation
Retrospective
754
7 years
11.4% of implantation
Retrospective
685
10.9 years
Retrospective
190
19 years
17.7% ( 15.4% of implantation)
Baker II-IV
62%
Retrospective
1529 (825 cosmetic;
264 reconstructive)
23.3 years
Spear et al., 2003
Camirand et al.,
1999
Seify et al., 2005
Cunningham et
al., 2007
Prospective
Retrospective
Prospective
Bengtson et al.,
2007
Prospective
Spear et al., 2007
Prospective
Kjoller et al.,
2002
Kulmala et al.,
2004
Holmich et al.,
2007
Handel et al.,
2006
85 cosmetic revisions
18
Average
follow-up
11.5 months
Capsular contracture
2% Baker II
No Baker III-IV
Baker III-IV:
1.8% cosmetic primary
augmentation
9.5% reconstructive
4.3% (Baker II-IV)
2.2% primary reconstruction
6% revisions-reconstruction
8.3% primary reconstruction
16.3% revisions-reconstruction
3 years
Baker III-IV:
5.9%
6 years
Baker III-IV:
14.8% primary augmentation
20.5% revisions-augmentation
15.9% primary reconstruction
Baker III-IV per 1000 patientmonth:
1.99 cosmetic
5.37 reconstructive
4.36 revision
Capsular
contracture
implantation[13],[14],[20],[58].
may
be
apparent
within
the
first
year
after
Breiting et al.[9] reported 18% of severe breast pain,
indicative of severe capsular contracture and in a previous study, involving a subgroup
of this population they had diagnosed 45% of capsular contracture (Baker II to IV) of
the breast after a 5-year period following breast implantation[96]. Capsular contracture
may also be symptomatic several years after surgery[9],[20],[58],[59]. The follow-up time
period remains, until now, unclear.
1.3 Estrogens
It is well known the protective role of estrogens in the progression of liver
fibrosis[97],[98] and the fact that estrogen deprivation has been associated with declining
dermal collagen content and impaired wound healing[99]. Nevertheless there are no
studies reporting menopause or estrogens versus capsular contracture. In this thesis
(Study 1) we purpose to analyse the association between capsular contracture and
menopause or estrogen status.
1.3.1 Estrogens review of the literature
By 1990s, there were estimates that up to 50% of postmenopausal women in
western Europe[100] and about 35% in United States[101] were on hormone replacement
therapy because of numerous beneficial effects attributed to such therapy[102],[103].
In 2002 the Women´s Health Initiative (WHI)[104] trial reported that combined
use of an estrogen and progestogen regimen increased the risk of breast cancer and
cardiovascular events and decreased the risks of fracture and colorectal cancer. Since
the publication of results from the WHI[104], many women have either stopped or
become reluctant to use hormone replacement therapy[104],[105] and clinicians have had to
19
revise their treatment algorithms. The relative risk (RR) to benefit ratio of hormone
replacement therapy was shifted toward excess risk by this study, and the use of
hormone replacement therapy after publication of this trial dramatically declined. A
significant minority of postmenopausal women remains on hormone replacement
therapy for treatment of menopausal symptoms, osteopenia, or personal preference.
The three most commonly used hormonal replacement therapy regimens are:
estrogen-only, continuous combined (estrogen and progesterone) and sequential
combined (estrogen followed by progesterone). In postmenopausal breast, the number
of estrogen receptors-positive cells within lobules is increased to about 50% in the
absence of hormone therapy[106]. In animal studies, long-term estrogen or combination
estrogen/progesterone therapy increases cell proliferation and the percentage of
glandular tissue in the breast[107],[108],[109] but the pathology of the breast with hormone
replacement therapy has not been well established[104],[110],[111],[112],[113],[114].
Staa et al.[115] reported that hormone therapy used for 5 years initiated at age 45
increased the absolute risk of myocardial infarction by 0.04% and breast cancer by 0.3%
and reduced the risk of hip fracture by 0.03%. In most of the younger hormone therapy
users, the frequency of risks exceeds that of the benefits, although the absolute excess
risks are small.
Even though estrogen therapy can significantly improve vasomotor symptoms,
postmenopausal Portuguese have a low rate of estrogen replacement therapy use, just as
surgically menopausal women in Taiwan[116].
20
1.4 The subclinical infection in the development of capsular contracture
Investigation of capsular contracture associated with breast implants has
focused on microorganisms found in the periprosthetic capsule or outer implant
surface[61],[62],[74],[76],[87],[117],[118],[119],[120],[121],[122],[123], inflammatory responses[81],[82],[124]
and histological characteristics of the capsule[44],[60],[84],[125],[126],[127],[128],[129].
There is evidence that bacterial colonization of mammary implants is partially
responsible for capsule contracture, and coagulase-negative Staphylococci, particularly
S.
epidermidis,
have
been
[71],[72],[130],[131],[132],[133],[134],[135],[136],[137],[138],[139],[140],[141],[142]
largely
implicated
. Adams et al.[62],[64] results
explained that S. epidermidis colonization of mammary implants is more likely to occur
because of bacterial contamination at the time of implantation than because of ongoing
contamination from the adjacent ductal system. Because of the low pathogenicity of
coagulase-negative Staphylococci and the existence of organisms in a dormant phase
within the biofilm around the implant, capsular contracture does not usually clinically
manifestate until some remote time after placement of mammary implants.
The authors perform microbial analysis of rabbits’ skin, operation air, capsules,
tissue expander and breast implants, to clarify the contamination surrounding the
procedure (Studies 2, 3, 4 and 5). In study 3, infection surrounding breast implants in
the presence of coagulase-negative Staphylococci was performed.
21
1.5 Histology and capsular pressure
Histologically, fibrous capsules showed three-layered composition (Figure 2):
- A inner layer abutting the silicone surface: single or multilayered formed basically by
macrophages (histiocytes) not with abundant fibroblast[124] .
- A thicker layer of collagen bundles arranged in a parallel array[59],[143] or
haphazard[144].
- A outer layer comprised loose or dense connective tissue with vascular supply[124] .
Figure 2. a) Inner layer; b) Middle layer c) Outer layer (hematoxylin-eosin stain
magnification 100x; from the Control group in Study 4)
a)
b)
c)
From a clinical perspective, most authors consider the degree of capsule
thicknesses to be commensurate with the severity of capsular contracture; this has never
been definitively proven as some reports found no correlation between microbiological
contamination, thickness and clinical contracture[66].
22
In publication I[84], on gross examination of the capsules, the Control group
capsule appeared more transparent and had less vessel predominance on the capsular
surface. The Fibrin group had a more opacified capsule and in many cases appeared
thicker. The average capsular thickness (histologically measured) was 0.6 mm in the
rabbit Control group, 1.0 mm in the rabbit Fibrin group and in human capsules, and 2.5
mm in human capsule contractures. There was a non–statistically significant increase in
capsular thickness in the Fibrin group. Hematoxylin and eosin sections of rabbit Control
capsules at 8 weeks, rabbit Fibrin capsules at 8 weeks, human capsules, and human
contractures were compared. Synovial-like reaction of fibrohistiocytic cells (synovial
metaplasia) was most pronounced in the rabbit Control capsule at 8 weeks, focal in the
rabbit Fibrin capsules at 8 weeks, and absent in the human contractures and control
capsules. The differences in synovial metaplasia in the specimens constitute a
histological detail that carries no clinicopathological significance; however, they were
reported for the sake of completeness. Inflammation (consisting of lymphocytes,
histiocytes, and eosinophils) was moderate in the 8-week rabbit Control capsule and
mild in the 8-week rabbit Fibrin capsule. The human capsule demonstrated minimal
inflammation, whereas the human contracture showed mild inflammation. The degree of
fibrosis was greater in the 8-week rabbit Fibrin capsules and human contracture than in
their counterparts (the 8-week rabbit Control and human capsules, respectively).
In the revision article from Broughton et al.[145] about wound healing, it is known
that early in the wound healing process the matrix is thin and compliant and allows
fibroblasts, neutrophils, lymphocytes and macrophages to easily maneuver through it; as
the matrix becomes denser with thicker, stronger collagen fibrils, it becomes stiff and
less compliant. Isometric tension is defined as a situation in which internal and external
23
mechanical forces are balanced such that cell contraction occurs without cell shortening
or lengthening which explains the higher pressure in capsule with contracture.
In Adams and Marisa et al.[84] (Publication I) the pressure-volume curve was
generated at 2 and 8 weeks. There was no significant difference between the Fibrin and
the Control groups at 2 weeks; however, at 8 weeks there was a significant increase in
intracapsular pressure in the Fibrin group. The limitation of this study was the
measurement of intracapsular pressure, given that it was not recorded directly but
through a small capsular window. The purpose of this study was to record directly the
pressure and to realize how fibrin modulates the capsule formation.
The underlying mechanism behind this process involves the activation of the
myofibroblast cells within the capsule, which supposes that contractile elements exert
the force necessary to produce capsular contracture. Myofibroblasts contain the
contractile elements actin and myosin and have been identified inconsistently within the
capsules of implanted devices; however, they have proven difficult to culture and study
in detail and, when found in the capsule, are found in exceedingly small quantities, are
located sporadically throughout the capsule, and are not found to attach to each other.
This scenario poses an inconsistent model for the development of contractile forces
necessary to produce contracture.
To study capsule firmness and the contracture development, we measured the
capsule pressure directly[146], reason why studies 2, 3 and 5 were performed with tissue
expanders.
Histological analysis of the capsule was performed in all studies.
24
1.6 The immunology of fibrosis
Fibrosis is an excessive extracellular matrix (ECM) due to the formation and
production cells and the occurrence of mononuclear inflammatory infiltrates, with
proliferation and activation of myofibroblasts. In this context, macrophages and mast
cells have been implicated as important participants in the inflammatory process
involving fibrosis.
Fibrosis is a major global health problem, but its etiology, pathogenesis,
diagnosis and therapy have yet not been addressed. Fibrosis can occur as a consequence
of many pathological conditions: 1) spontaneous (keloids, Dupuytren´s contracture); 2)
from tissue damage (post-operative adhesions, burns, alcoholic and post-infection liver
fibrosis, silica dust, asbestos, antibiotic bleomycin); 3) inflammatory disease (infections,
scleroderma); 4) in response to foreign implants (breast implants, cardiac pacemakers,
heart valves, artificial joints, central venous catheter ports); and 5) from tumors
(fibromas, neurofibromatosis).
Several mutually non-exclusive hypotheses have been proposed: 1) infection; 2)
reaction to altered self; 3) overproduction of reactive oxygen species (ROS) and nitric
oxide (NO); and 4) mechanical stress.
In all cases studied, the early stages of fibrotic conditions are characterized by a
perivascular infiltration of mononuclear cells and the subsequent imbalance of anti and
profibrotic cytokine profiles. One of the most prominent activators of mononuclear cells
and fibroblasts are hyaluron fragments that not only induce the expression of various
cytokines (IL-1, IL-12 and TNF-α), chemokines (MPI-1A, MCP-1, IL-8) and inducible
nitric oxide synthase (iNOS), but also trigger the expression and secretion of
macrophage-derivated matrix metalloproteinases (MMP), enzymes essential for ECM
cleavage[147].
25
Mast cells can play a role in fibrosis by their secretion of tryptases, contributing
to connective tissue breakdown. As a consequence of activation of procollagenase and
induction of a cascade of MMPs, the connective tissue becomes more penetrable for
infiltrating leucocytes during inflammation. Mast cell-derived tryptase indirectly
induces fibroblasts proliferation by stimulating the synthesis of cyclooxygenase and
prostaglandins[148],[149]. Natural killer (NK) cells display predominantly anti-fibrotic
properties in several fibrosis model systems[150]. Furthermore, NKT-derived interferon
(IFN)-y inhibits the production of the profibrotic cytokine transforming growth factor
beta (TGF-β1)[151].
Cells and cytokines play a prominent role in the initiation and progression to
fibrosis and Th1 and Th2 cytokines play opposing roles in fibrosis[152]:
- Th1 cytokines (IFN-y and IL-12) suppress the development of tissue fibrosis.
- Th2 cytokines (IL-4 and IL-13) are strongly pro-fibrotic.
Fibroblasts can be derived from local quiescent connective tissue fibroblasts by
proliferation, but there is also ample evidence that at least some of them originate from
myeloid precursors in the blood or bone marrow that then migrate to sites of injury[153].
Once in an active state, fibroblasts are designated as myofibroblasts which express αsmooth muscle cell actin (α-SMA), produce increased amounts of ECM proteins, such
as collagen type I and fibronectin, proliferate and show contractile properties. Their
usual activators are IL-6 and TGF-β1, although they can also be activated by a variety
of other cytokines, chemokines, growth factors, components of microbial cells walls
and members of oxidative burns cascade[152]. Fibroblasts also receive stimuli from
lymphocytes via the CD40-CD40 ligand (CD40L or CD154); CD40 ligation results in
the synthesis of IL-6, IL-8, hyaluronan and the adhesion molecules ICAM-1 and
VCAM-1[154]. Among the various pro-and anti-fibrotic cytokines, TGF-β isoforms seem
26
to play a key role in the development of fibrosis[155],[156]. TGF-β1 has a fibrogenic role
while TGF-β3 has anti-fibrotic properties. Studies on the role of TGF-β2 are rare and
the results contradictory. TGF-β1 is a central mediator of fibrosis, but alone it is
insufficient to cause a persistent fibrotic response; only in synergy with other profibrotic cytokines, such as connective tissue growth factor (CTGF), results in chronic
fibrosis[157].
In summary TGF-β1, CTGF, osteopontin (OPN), IL-4, IL-6, IL-10, IL-13, IL21, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin like
growth factor-1 (IGF-1), platelet-derived growth factor ( PDGF), oncostatin M and
endothelin 1 (ET-1)[158] all promote fibrosis , whereas IFN-y, TGF-β3, IL-10 and IL-12
are anti-fibrotic. IL-5[152], TGF-β2 and TNF-α[159], exerting either pro-or anti-fibrotic
activities depending on the disease, animal model and experimental settings.
1.6.1 Pathophysiological hallmarks of breast implant capsule formation
- Fibroblasts and macrophages (by its location in connective tissue namely histiocytes),
formed a palisade-like multilayered cell wall toward the silicone implant, and represents
the major cell population[124] .
- Ample presence of T cells (CD4+/CD8+), macrophages, dendritic cells (DCs), CD25
and CD45RO expressing cells; Langerhans-cell like denditric cells are found at the
frontier layer zone abutting the silicone implant[59],[124],[125] .
- No accumulation of B-cells[59],[124],[125].
- Cells at the frontier layer, endothelial cells and smooth muscle cells showed massive
HSP60 expression (reflecting the mechanical effect or other forms of stress exerted on
implant and capsule); HSP60 positively predominantly in fibroblast, followed by
macrophages and T-cells[124].
27
- The layers in closest proximity to the silicone showed massive expression of adhesion
molecules namely intercellular adhesion molecule (ICAM-1) but not to E-selectine or
VCAM-1; the endothelial cells of the neovasculative vessels in the fibrous capsules
were P-selectine positive[124].
- Actin+ smooth muscle cells found in vascular walls but also in interstitium,
occasionally formed dense bands[124].
- Collagenous extracellular matrix (ECM) proteins: high procollagen (type I and III)
expression correlated with high fibrotic activity; the proportion of procollagen to
collagen, showed a decreased in procollagen expression and an increase of mature
collagen deposition with longer implant duration[124].
- Non-collagenous extracellular matrix (ECM) proteins: fibronectin shows a high
affinity for silicone and for cellular components such as macrophages, fibroblasts and
T-cells; tenascin, mainly synthesized for fibroblasts, mediate adhesion of mononuclear
cells in on the frontier zone[124].
- Serum proteins from many protein families adhere to silicone surface and mediate
adhesion of fibroblasts, macrophages and ECM proteins[160].
- Macrophages are activated by cryptic or altered protein domains exposed on silicone
surfaces or by silicone degradation products[161].
- Activated intracapsular lymphoid cells stimulate transdifferentiation of fibroblasts to
myofibroblasts by CTGF, IL-1 and TNF-α. Macrophages contribute to this process by
the production of TGF-β1 and IL-6[162].
- Soluble ICAM-1, procollagen III, circulating immune complexes and anti-polymer
antibodies are elevated in sera of women with strong fibrotic reactions to silicone[163] .
28
- A special ELISA-based system (SILISA®) demonstrating the “signature” of serum
protein adhesion to different silicone types can be used to determine the potential risk of
fibrosis development around silicone breast implants[164].
Summary:
- The immune response comprises primarily T-cells.
- The preferential distribution of dendritic cells in the frontier layer zone underlines that
this immunological process is not identical or comparable with an unspecific local
immune reaction or so called foreign body granuloma formation.
- The constant presence of CD1a+ cells in the frontier zone adjacent to the silicone
implant as well as next accumulation of CD4+ cells support the hypothesis that silicone
is not inert, as postulated by the manufacturers, but induces directly or indirectly a Tcell immune response. The peri-implant connective tissue capsule may represent a
possible site of antigen processing and presentation[163].
- The massive deposition of tenascin in the frontier layer zone supports the theory of
mechanical stress depending of tenascin expression[163]. T-lymphocytes significantly
increase the synthesis rate of tenascin via certain cytokines such as IL-4 and TNF-α[165].
- The mechanical stress to which breast implant is exposed is associated with HSP60
expression, a family of highly conserved proteins produced by all cells in response to
various physiological and non-physiological stress-situations to protect the cells from
potential lethal assaults[163]; HSP70 was associated with structural changes of the
implant capsule, in terms of capsular thickness and the Baker score[166].
29
1.6.2 Microdialysis, IL-8 and TNF-α
Microdialysis enables measurement of the chemistry of the capsule extracellular
fluid. Although initially developed over 30 years ago[167], microdialysis studies in
humans have been mainly limited to head injury[168],[169],[170],[171], subarachnoid
haemorrahage[172], epilepsy[173] and cerebral tumors[174],[175].
The chemotatic cytokine (chemokin) IL-8 (CXCL8) is an important mediator in
pathogenesis of many acute and chronic inflammatory disorders[176]. IL-8 mainly targets
polymorphonuclear cells (PMN), the major phagocyte cell, but also mediates attraction
of basophils, eosinophils and T-cells to the inflammatory site[177].
Interleukin-8 (IL-8) is induced by a wide range of stimuli, including: TNF-α, IL1[178],
bacterial
agents[179],
formyl-methionyl-leucyl-phenylalanine
(f-MLP)[180],
zymosan[181], plated factor 4 (PF-4)[182], and P-selectin together with RANTES
(regulated upon activation normal T-cell expressed presumed secreted)[183]. The many
cell types thus responding are: monocytes[184], PMN[185], endothelial cells[186],
fibroblasts[187], T-lymphocytes[188], natural killer cells (NK)[182] and human mast cell line
[189]
. In the study by Lund et al.[190], lipopolysaccharide (LPS), a component of the outer
membrane of Gram-negative bacteria, potentially induced IL-8 release in monocytes,
while TNF-α was a good inducter of IL-8 in PMN. Furthermore, a relatively high level
of IL-8 was associated with PMN cells. Lund et al.[190] concluded that under
pathophysiological condition-associated exposure of blood to LPS, one may anticipate
that IL-8 is generated as a direct effect of LPS acting on monocytes and that it is further
amplified due to TNF-α endogenously produced by monocytes.
IL-8 is an important chemotactic regulator of neutrophil in vivo[177], and its
concentration increases during different infections, such as bacteraemia[191] and
meningococcal infection[192]. IL-8 concentrations have also been demonstrated to play
30
an important role in the immunological response to inflammatory disorders
characterized by neuthrophilic infiltration including psoriasis[193], rheumatoid arthritis
and asthma.
To monitor levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α),
the authors utilized microdialysis, and to our knowledge, this had never been previously
studied in capsule extracellular fluid by this technically demanding method.
1.7 Prevention and treatment of capsular contracture
Despite innovations in shell surface textures, implant shapes, inner gel
composition,
surgical
implantation
techniques
and
pocket
irrigation[20],[38],[39],[55],[62],[63],[64],[194],[195],[196],[197],[198],[199],[200],[201],[202],[203],[204],[205]
to
prevent capsular contracture, this major complication remains a serious problem.
In a pre-clinical study by Tamboto et al.[206], the authors concluded that
Staphylococcus epidermidis biofilm formation was associated with a fourfold increased
risk of developing CC. To prevent CC, many plastic surgeons follow the general
principles of the “Betadine Era”[62] and the “Post-Betadine Era”[64],[87]. However, it also
known that other factors related to wound healing influence the development of this
clinical condition[124]. In preclinical studies, the treatment with mesna[126], mitomicina
C[207], zafirlukast[208],[209],
pirfenidone[210] or halofuginone[211] reduced capsule
thickness, fibroblast cell proliferation and collagen deposition. Nevertheless, these
drugs are not commonly used in clinical practice, with the exception of the the
antileukotriene
drugs
(zafirlukast,
montelukast
and
pranlukast).
Scuderi
et
al.[212],[213],[214] reported clinical experience with zafirlukast and suggests that this drug
may be effective in reducing pain and breast capsule distortion in patients with
longstanding contracture who are either not surgical candidates or who do not wish to
31
undergo surgery. The antileukotriene drugs are currently used in asthma and lung
diseases, however, the experience is limited to severe CC because of the severity of
possible side effects such as liver failure[215] or Churg-Strauss syndrome[216]. Research
concerning cause and prevention has moved forward; however, in clinical practice is
still a difficult issue, especially when comparing decreased CC rates achieved with
polyurethane implants.
Some reports correlate clinical contracture and hematoma[16],[60]; to clarify this
implication, the authors perform study 2 with tissue expanders surrounded by rabbit´s
blood to simulate a hematoma, and tissue expanders in the presence of thrombin
(FloSeal®), an absorbable hemostatic agent and in the presence of a fibrin wound
healing agent (Tisseel/Tissucol®).
Recent evidence investigating the chitosan and the chitooligosaccharide, have
revealed that they have intrinsic antibacterial and antifungal activity[217],[218],[219],[220] and
ability to bind growth factors[221]. In study 4, breast implants impregnated with
chitooligosaccharide mixture (COS) and low molecular weight chitosan (LMWC) were
introduced in the rabbit model.
Steroids have shown to be effective in treatment of others pathologic disorders
characterized for an unorganized scar tissue in dermal structures[222],[223],[224],[225], as
keloids , hypertrophic scars and burn scar contractures[226]. Corticosteroids administered
during wound healing showed to stop the growth of granulation completely, the
proliferation of fibroblasts, diminish the new outgrowths of endothelial buds from blood
vessels and stop the maturation of the fibroblasts already present in connective
tissue[227].
Also when administered early after injury, corticosteroid delay the
appearance of inflammatory cells, fibroblasts, the deposition of ground substance,
collagen, regenerating capillaries, contraction, and epithelial migration[228]. These data
32
raised the interest on the use of steroids in the treatment and prevention of CC. The data
available in the literature regarding the effect of steroids in the prevention and treatment
of CC is spare and contradictory. The steroids have an important role in the earlier
phases of wound healing[228], and the role of those effects on the early phase of breast
capsule formation are also not understood nor explored. In study 5, breast implants with
triamcinolone were introduced in the rabbit model.
1.7.1 Tissucol/Tisseel®
Fibrin glue consists of two components, a fibrinogen solution and a thrombin
solution rich in calcium. Fibrin serves as a binding reservoir for several growth factors
such as vascular endothelial growth factor (VEGF)
[230]
[229]
, transforming growth factor-β1
and basic fibroblastic growth factor (bFGF)[231]. Fibrin glue has been studied for
decades for its use surgically as a hemostatic and sealant agent. It is routinely used in:
gastrointestinal anastomosis, breast surgery, face-lifts, abdominoplasty, nerve repairs,
graft
securing,
neurosurgery
[232],[233],[234],[235],[236],[237],[238],[239],[240],[241],[242]
and
ophthalmology
. More recently it has also gained attention
as a possible means to deliver drug therapies[243]. For example, in a study by Zhibo and
Miabo[244], release of lidocaine from fibrin glue for pain reduction was tested in humans
after breast augmentation. Patients who received fibrin glue with lidocaine in the
subpectoral pocket experienced less pain than those who received the same amount of
lidocaine or fibrin glue alone.
To study the implications of wound healing in development of capsular
contracture, the instillation of fibrin (Tissucol/Tisseel®) in the implant pocket, to induce
hemostasis and as a tissue glue to bind the tissues together (adhesive properties), was
performed (Studies 2 and 3); numerous reports have demonstrated that fibrin glue
33
application is an effective adhesive that is associated with improved parameters of
wound healing[245],[246],[247],[248]. In Adams and Marisa et al.[84] (Publication I), we have
demonstrated exactly the opposite; in this study, one experimental group has been
instilled with 5 cc of fibrin glue [fibrin glue is prepared with 4 ml of rabbit cryo (PelFreez; Pel-Freez Biologicals, Rogers, Ark.), 500_l of 10% CaCl (Sigma- Tau
Pharmaceuticals,
Gaithersburg,
Md.),
1000
units
of
thrombin
(Monarch
Pharmaceuticals, Bristol, Tenn.) in 1 ml of 50 mM TrisCl (Sigma), pH 7.4] into the
implant pocket as a contracture inducing agent. Even if there was a non–statistically
significant increase in capsular thickness in the Fibrin group, the degree of fibrosis was
greater in the 8-week rabbit fibrin capsules and human contracture than in their
counterparts (the 8-week rabbit control and human capsules, respectively). The purpose
of this study is to clarify the impact of fibrin in contracture development. Incidentally,
studies 1 and 2 were performed with fibrin (Tissucol/Tisseel®), to induce hemostasis
and as a tissue glue to bind the tissues together (adhesive properties), which is different
from the one used in publication I. As indicated by Sead et al.[249], fibrin sealant
prepared from Tisseel kit without aprotinin has the ability to reduce extracellular matrix
and TGF-β1 mRNA levels, especially from adhesion fibroblasts, which may indicate a
role in reduction of postoperative adhesion development. As it has been demonstrated,
TGF-β is a mediator in scar formation and in multiple fibrotic disorders. It has also been
demonstrated that connective tissue growth factor (CTGF) is a downstream mediator of
TGF-β and acts to stimulate wound contraction and fibrosis. It has been observed that
local treatment with antagonists/anti-sense-oligonuceotides of TGF-β and CTGF at the
time of surgery reduced CTGF levels in tissue and correlated with reduced capsular
formation in a rat model. The study by Cole et al.[250] supports the use of fibrin to
deliver MALP-2 and possibly other peptides, in an active form that might enhance
34
wound healing. In the increase understanding of the wound healing process, it becomes
clear to Brissett et al.[251], that cellular recruitment and release of growth factors are
paramount for normal healing to occur; a delay in this process can result in a chronic
wound or excessive scar. Although the use of these preparatins (Tisseel and Vi-Guard)
allows the closure of dead-space and approximation of the skin flaps, it is argued that
these tissue adhesives produce such a dense architecture that angiogenesis and vascular
ingrowth are inhibited; in addition, because these tissue adhesive do not possess growth
factors or cytokines to actively recruit cells that are essential for wound healing, they
are considered bioactively inert. The study by Petter-Puchner et al.[252] was designed to
assess the impact of fibrin sealing with Tissucol/Tisseel® on adhesion formation to
condensed polytetrafluoroethylene meshes as well as on tissue integration of these
implants in experimental intra-abdominal peritoneal on lay mesh repair in rats. The
authors concluded that Tissucol/Tisseel® improves the tissue integration and reduces
early adhesion.
1.7.2 FloSeal®
FloSeal® does not contain any fibrinogen (different from the above fibrin
sealant); it requires blood as a source for fibrinogen, for clot activation and is ineffective
in the absence of any bleeding. FloSeal® is a combination of a gelatin-based matrix
from bovine collagen containing microgranules, cross-linked with glutaraldehyde and
human thrombin solution[253]. Upon contact with blood the gelatin particles swell and
induce a tamponade-like effect. This characteristic allows it to be effective in
controlling moderate arterial bleeding. Numerous reports have demonstrated that
FloSeal®
successfully
reduces
bleeding
in
cardiac
surgery[254],
urologic
procedures[255],[256],[257], gynecology[258],[259] and neurosurgery[260]. Dogulu et al. [261], in a
35
pre-clinical model, concluded that the application of FloSeal® at a laminectomy site
may be useful to decrease adhesion at the interface between the dura mater and epidural
fibrosis.
1.7.3 Triamcinolone acetonide
The data available in the literature regarding the effect of steroids in the
prevention and treatment of CC is spare and contradictory. Perrin[262] reported less than
5 percent of significant capsule formation on patients submitted to augmentation
mammaplasty with inflatable breast prostheses filled with saline and a cortisone
derivative, with no evidence of wound complications attributable to the steroid. This
results were reinforced by Ksander[263] in a pre-clinical model with rats, where it was
reported that saline implants filed with saline solution were harder and surrounded by a
thicker capsular membrane than those filed with metilprednisolone sodium succinate, at
60 and 120 days. Caffee et al.[264] reported in a preclinical study, that triamcinolone in
the pocket during surgery was ineffective for prevention of capsular contracture, but if
injected 4 and 8 weeks postoperatively, the drug was able to completely eliminate
contracture. Caffee et al.[264] assume that triamcinolone in the pocket was not effective
because its effect does not last long enough, and the objection to this method has been
the fact that the drug was given at the time of operation and was therefore most effective
in the early phases of wound healing and less active in the latter stages when contracture
is more likely to begin. However, betadine[62] and antibiotic breast irrigation[64],[87] were
clinically associated with a low incidence of CC and more effective in the early phases
of wound healing and less active in the latter stages.
The majority of patients
undergoing breast augmentation will never experience contracture, and therefore, it did
not seem reasonable to apply an experimental invasive method to such a group only a
36
minority of patients who would potentially benefit. Caffee et al.[264] conclusions were
based on indantation and applanation tanometry. There have been no further reports
confirming that triamcinolone in the pocket during surgery was ineffective. Morover,
Caffee et al. in 2002[265], and Sconfienza et al. in 2011[266], reported clinical success
treating patients with CC with the injection of triamcinolone-acetonide between the
capsule and the implant. Derendorf et al.[267] reported a plasma half-life after venous
injection of 2h. Recently, Yilmaz et al.[268] performed an extensive review of human and
experimental studies published on the pharmacokinetics of TA for the treatment of
macular edema. The authors concluded that the pharmacokinetic profile of TA is
unpredictable and the agent has a time-limited therapeutic action due to its relatively
short half-life. This has led to the need for repeated injections to treat contracture or
macular edema. The answer to the clinical efficiency of triamcinolone-acetonide with
various doses is not known.
1.8. Chitosan and Chitooligosaccharides
Chitin, the polymer D-glucosamine in β (1,4) linkage, is the major component
of exoskeleton of crustaceous and cell wall fungi[269]. Chitosan (CS) is the deacetylated
product of chitin. Chitooligosaccharides (COS) are degraded products of chitosan, or
the deacetylated and degraded products of chitin, by chemical and enzymatic
hydrolysis. In the literature, the term chitosan is used to describe chitosan polymers
with different molecular weight (50-2000 kDa), viscosity and degree of deacetilation
(40-98%)[270].
Material
with
lower
levels
of
deacetylation
degrades
more
rapidly[271],[272],[273]. Chitosan has been the better researched version of the biopolymer
because of its ready solubility in dilute acids rendering it more accessible for utilization
and chemical reactions[274].
37
Chitosan and related chitooligosaccharides have intrinsic antibacterial and
antifungal activities[217],[218],[219],[220], which permit the study of the infectious hypothesis.
On other hand, its ability to bind to growth factors[221],[275], the hemostatic action[276], the
ability to activate macrophages and cause cytokine stimulation[276] and to increase the
production of TGF-β[277] allows the study of the hypertrophic scar hypothesis.
Chitosan can be processed in a variety of different shapes. These attributes make
chitosan a promising biopolymer for tissue engineering due to its excellent
biocompatibility. Chitosan applications include use in wound healing (full thickness
skin defect, dermal burns)[218],[221],[278],[279], in target delivery of low molecular drugs[280],
in orthopaedics (cartilage, anterior cruciate ligament, intervertebral disc, bone,
osteomyelitis )[220],[281], in otologic diseases (tympanoplasty)[279] and in breast capsular
contracture[282]. The combination of chitosan with materials is common in various
reports[274]. The results published by Khor et al.[274], from cell line culture and animal
model studies, indicated that chitin and chitosan materials were non-cytotoxic and
suggest that these materials would provide tissue engineered implants that are
biocompatible and viable. Baldrick et al.[276] observed that chitosan has local biological
activity in the form of hemostatic action and, together with its ability to activate
macrophages and cause cytokine stimulation (which has resulted in interest in medical
device and wound healing applications), may result in a more careful assessment of its
safety as a parenteral excipient.
Literature data reporting general toxicity testing for chitosan is limited[276]. An
investigation of intestinal absorption of chitosan in rats showed that the material
underwent digestion into low molecular weight substances within the gastrointestinal
tract, and that they are distributed extensively in tissues[283]. Apparent toxicity was seen
with 653-720 mg/Kg/day of COS in rats with side effects in skin and fur and decrease
38
bodyweight[284]; it is further suggested that increased platelet count, lymphocyte count
and differential neutrophils count may be related to dermal inflammation. High dose
effects were also seen in rabbits following intravenous dosing of chitosan, with deaths
at 50 mg/Kg/day (but no effect at 4.5 mg/Kg/day)[285]; it was suggested that the finding
was probably due to cell aggregation. Studies in dogs[286] showed evidence of toxicity
following subcutaneous dosing with clinical signs (anorexia) from 30 mg/Kg/day,
chemistry changes (especially neutrophilia) from 50 mg/Kg/day, and severe dyspneia
and deaths from 150 mg/Kg/day; pathological examination showed severe pneumonia
in the latter animal and it was suggested that this finding was possibly induced by
immunological reaction and cytokine activation. Cytotoxicity was demonstrated with an
inhibitory concentration (IC50) of 0.2 mg/ml for chitosan hydrochlorid with release of
haemoglobin, damage of the erythrocyte membrane, cell aggregation and complete
lysis[285]. Intratumoral injection of chitosan on tumor bearing mice, increases the rate of
tumor growth, metastasis and the number of capillaries formed[287]. There were no
reports in rabbits related with impregnated chitosan breast implants or with toxicity
after chitosan implantation.
1.9 The New Zealand white rabbit
Adams and Marques et al.[84] (Publication I) reported a model to study capsule:
the New Zealand white rabbit. The New Zealand white rabbit has the capability to
support tissue expanders and breast implants, which is impossible in mice; porcine had
limited reports.
This thesis is an extension of Adams and Marques et al.[84] study (Publication I).
In this study New Zealand white rabbits (n = 32) were subdivided into experimental (n
= 16) and control groups (n = 16). Each subgroup underwent placement of smooth
39
saline mini implants (30 cc). The experimental group underwent instillation of fibrin
glue into the implant pocket as a capsular contracture-inducing agent. Rabbits were
euthanized from 2 to 8 weeks after the procedure. Before the animals were euthanized,
each implant was serially inflated with saline and a pressure-volume curve was
developed using a Stryker® device to assess the degree of contracture. Representative
capsule samples were collected and histologically examined. Normal and contracted
human capsular tissue samples were also collected from patients undergoing breast
implant revision and replacement procedures. Tissue samples were assessed
histologically. Pressure-volume curves demonstrated a statistically significant increase
in intracapsular pressure in the Fibrin group compared with the Control group. The
Fibrin group had thicker, less transparent capsules than the Control group. Histological
evaluation of the rabbit capsule was similar to that of the human capsule/contracture for
the Control and the Fibrin groups. The authors concluded that pathological capsular
contracture can be reliably induced in the rabbit. This animal model provides the
framework for future investigations testing the effects of various systemic or local
agents on reduction of capsular contracture.
In the discussion of this paper (Publication I) performed by Burkhardt[84], the
author believe that if a rabbit model must be used for research, a more appropriate
model is that reported by Shah et al.[288],[77], who used bacterial contamination to
produce contracture. In opposite to our belief that the cause of contracture is
multifactorial, to include hematoma, granuloma, foreign body reaction, hereditary
factors and subclinical infection as any one of these factors may theoretically stimulate
an internal hypertrophic scar response that then becomes a contracted capsule,
Burkhardt believes that presumed cause is limited to infection or bacterial
contamination.
40
The end result is that the histological analysis of the rabbit fibrin model was
similar to human contracture but the limitation of this study was the inability to provide
a clinical translation of this contracture model, as no rabbit developed a Baker II, III or
IV. Moreover, this was a pilot study, and the fibrin modeling response in capsule
formation deserves further studies.
This thesis is an extension of the Adams and Marques et al.[84] study (Publication I)
to clarify the etiology of capsular contracture, based on this animal model, and with the
hope of developing a clinical capsular contracture model.
1.10 The pig and the mice models
Two recent studies introduced a pre-clinical CC model; 1) Tamboto et al.[206]
developed a pig model of CC with submammary pockets inoculated with S. epidermidis
before miniature gel-filled implants introduction; 2) Katzel et al.[289] developed a mice
model implanted with silicone gel implants then received a 10-Gy directed radiation
dose from a slit-beam cesium source. These models brought to the science the
possibility of further promissory studies.
41
42
2. Aims of the thesis

Retrospective study in aesthetic and reconstructive groups of Portuguese women
who received silicone textured breast implants within 1998 to 2004. Report the
occurrence and severity of postoperative complications focused on capsular
contracture. Analyse the impact of the follow up period, the Baker grade II subjects
and factors that might contribute to the development of capsular contracture rates,
namely estrogens and menopausal status (STUDY 1)

Identify bacteria and fungi from operation air, rabbit’s skin, tissue expanders, breast
implants and removed capsules (STUDIES 2, 3, 4 and 5)

Histological analysis of the capsule (STUDIES 2, 3, 4 and 5)

Monitor the levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in
capsule extracellular fluid by microdialysis (STUDY 4 and 5)

Identify the impact of hematoma in capsular contracture (STUDY 2)

Identify the impact of coagulase-negative Staphylococci in capsular contracture
(STUDY 3)

Identify the impact of thrombin (FloSeal®) in capsular contracture (STUDY 2)

Identify the impact of fibrin (Tissucol/Tisseel®) in capsular contracture (STUDIES
2 and 3)

Identify the impact of chitosan in capsular contracture (STUDY 4)

Identify the impact of triamcinolone acetonide in capsular contracture (STUDY 5)

Clarify the etiology of capsular contracture (STUDIES 2, 3 and 4)
43
44
3. Material and methods
STUDY 1
Subjects and data collection
Existing medical records of women who had undergone breast implantation with
customized textured silicone breast implants (Allergan, Santa Barbara, California, USA)
between 1998 and 2004 in the Hospital of S. João (Oporto, Portugal) were examined. A
total of 224 women were identified with 104 women who underwent cosmetic breast
augmentation (Cosmetic group) and 120 women who underwent postmastectomy
reconstruction of the breast (Reconstructive group).
The following data were collected from medical records: patient demographics,
alcohol and medication habits, medical history, surgical procedures, incision location,
implant device placement[290] and postoperative acute complications (hematoma,
infection, and seroma). Postoperative chronic complication data (capsular contracture,
folds, wrinkles, breast pain, and change of tactile sense) were not gathered from medical
records. Self-reported complications related to satisfaction with implantation surgery
were collected using a self-administered questionnaire. Women who answered the
questionnaire were asked to attend a consultation to be further evaluated by two trained
plastic surgeons in order to decrease subjectivity of this evaluation. The degree of late
capsular contracture was assigned by the plastic surgeons according to Baker’s
classification[25].
Women from the initial group (157 of 224) completed the self-questionnaire and
attended the consultation. The remaining 67 were then excluded (n = 35 women,
Cosmetic group; n = 32, Reconstructive group) to remove any potential bias that might
result from patients with incomplete data. Women were excluded due to loss of contact
45
as they moved out of Oporto or if no current mailing address or phone numbers were
available at the time of the study. The Reconstructive group was comprised of 88
patients with 115 breast implants with 27 patients having received bilateral breast
implants. The Cosmetic group had 69 patients with 136 breast implants from which 2
had a tuberous breast deformity, 1 had a unilateral aplasia and 1 had a Poland’s
syndrome. All cosmetic patients younger than 18 years old (n = 4) received implants
following medical indication, namely severe asymmetry, aplasia of breast tissue or
congenital malformation.
Statistical analysis
Postoperative local complications were analyzed independently for the entire
study group and individual clinical treatment groups and reported per woman and per
implantation operation (SPSS, Statistical Package for Social Sciences).
Possible
associations among recorded data sets of patients characteristics, surgical procedures
and complications were evaluated using Pearson 2 testing and logistic regression
modeling[291]. Trend analysis was performed using Chisquared Automatic Interaction
Detection (CHAID) method (SPSS, Statistical Package for Social Sciences, Chicago,
IL)[292], using the likelihood ratio chi-square statistic as growing criteria, along with the
Bonferroni 0.05 adjustment of probabilities, and setting the minimum size for parent
and child nodes at 10 and 5, respectively. Relative risks (RR) and 95 percent confidence
intervals (CI) were calculated for identified characteristics of interest to examine
strength and precision of statistical associations.
CHAID has not been widely applied to trend analyses in plastic surgery
investigations, but CHAID is one of the oldest tree-classification methods originally
proposed by Kass[292]. In brief, CHAID is an exploratory method to examine
46
relationships between a dependent variable (e.g. capsular contracture) and a series of
predictor variables (e.g.: type of cohort, age at surgery, follow up period, etc.) and their
interactions. The CHAID algorithm creates adjustment cells by splitting a data set
progressively via a classification tree structure where the most important predictor
variables are chosen that to maximize a chi-square criterion. The most significant
predictors defined the first split or the first branching of the tree. Progressive splits from
the initial variables resulted in smaller and smaller branches. The result at the end of the
tree building process is a series of groups that are different from one another on the
dependent variable. Classification trees lend themselves to be displayed graphically and
are far easier to interpret than numerical interpretation from tables.
STUDY 2
Eighteen (n = 18) New Zealand white female rabbits (3-4 kg) were implanted in
an approved institutional animal care protocol, with textured saline tissue expanders (20
ml, Allergan, Santa Barbara, California, USA) with intact connecting tube and port.
Prior to surgery the rabbit´s skin was washed with Betadine® Surgical Scrub containing
7.5% povidone-iodine, followed by Betadine® Solution containing 10% povidoneiodine (Purdue Products, Stamford, USA). The surgical procedure was performed in an
animal operating theatre following aseptic rules. Penicillin G 40.000 u/Kg IM was
administered just intraoperatively. Talc-free gloves were used at all times during the
procedure. Pockets were developed in the sub-panniculus carnosis along the back
region, with atraumatic dissection. Particular attention was given to hemostasis, under
direct vision avoiding blunt instrumentation and there was no obvious bleeding. A new
pair of talc-free gloves was used before tissue expanders insertion with minimal skin
47
contact.
Each tissue expander was placed and filled up to 20 mls volume. Four
expanders were placed per rabbit.
In each rabbit, 1 control and 3 experimental tissue expanders were placed. The
experimental groups were: 1) sprayed with 1 ml of fibrin glue (Tisseel/Tissucol®;
Baxter Healthcare Corporation, Vienna, Austria, Europe); 2) instillation of 2 ml of
rabbit´s blood into the expander pocket to simulate a hematoma; 3) instillation of 5 ml
of thrombin sealant (FloSeal®; Baxter Healthcare Corporation, Vienna, Austria,
Europe) into the expander pocket.
A pressure measure device (Stryker® instruments, Michigan, USA) was
connected to the tissue expander port and intra-expander pre-surgical pressure was
recorded directly. Pressures were recorded after each 5 ml increments until tissue
expanders were overfilled.
Rabbits were sacrificed at 2 or 4 weeks. Prior to sacrifice, each animal was
anesthetized and the dorsal back area shaved. The pressure measure device was
connected to the tissue expander port and intracapsular pressures were recorded 5 ml
increments previously to any incision in the capsule. Capsule samples were submitted
for histological and microbiological evaluation.
Microbiological Assessments

Air
Operating room air samples (n = 36) were collected during all surgical procedures
using the MAS 100-Eco® air sampler 00109227.0001 / 26299 at a flow rate of 100
L/min. Identification of bacterial and fungal isolates followed standard microbiological
procedures. Gram positive cocci were characterized by biochemical methods. Catalasepositive and coagulase-positive isolates were reported as Staphylococcus aureus;
48
catalase-positive and coagulase-negative isolates were reported as coagulase-negative
Staphylococci. Gram negative bacilli were characterized using the Vitek Two® with
version VT2-R04.02 software. Fungi were characterized according to the macroscopic
appearance and microscopic morphology.

Rabbit skin
A total of 54 contact plates (18 brain-heart agar, 18 mannitol salt agar and 18
Sabouraud agar contact plates) were pressed to the shaved dorsal skin surfaces. Brainheart and mannitol salt agar plates were incubated for 3 days at 28ºC; Sabouraud contact
plates were incubated for 7 days at 28ºC. The identification of the bacteria and fungi
followed the procedures reported above.

Capsules and tissue expanders
Excised implants and representative capsule samples were incubated at 37ºC for 3
days in brain-heart broth and examined daily; changes in turbidity of the broth media
were considered positive and were subcultured in solid agar media. Characterization of
microbial isolates followed the above described procedures.
Histological Assessment
Capsule specimens were fixed with 10% buffered formalin and embedded in
paraffin. Sections were stained with hematoxylin and eosin and evaluated histologically
for tissue inflammation and capsular thickness. The type of inflammatory cells was
grouped into 3 categories: 1) mononuclear (lymphocytes, plasmocytes and histiocytes);
2) mixed (mononuclear cells and eosinophils); and 3) polymorph (eosinophils and
heterophils/neutrophils). Inflammatory infiltrate intensity was categorized according to
the following criteria: absent (-); mild (+); moderate (++); and severe (+++)[125].
49
Samples were stained with Masson`s trichrome[293],[294] to characterize the
connective tissue (loose or dense), organization of the collagen fibers (arranged in a
parallel array or haphazard), angiogenesis (absent, mild, moderate or high) and fusiform
cells density (mild, moderate or high) were observed. The dense connective tissue was
semiquantitative analysed: a) dense ≤ 25%, thick collagen bundles less than 25%; b)
dense 25-50%; c) dense 50-75%; e) dense >75%.
Statistical analysis
Data was grouped according to the type of product applied to the tissue
expander, as none (Control), blood (Blood), Tissucol/Tisseel® (Fibrin) and FloSeal®
(Thrombin), and analyzed separately for rabbits sacrificed at 2 or 4 weeks after surgery
as well as for all 18 sacrificed rabbits. One-way analysis of variance was used to
compare the intra-expander pressure prior to insertion. A two-tailed paired t-test and the
nonparametric alternative Wilcoxon signed rank test were used to determine if
continuous variables (intracapsular pressure and histological measured thickness) were
significantly different between Control and experimental groups. Categorical variables
were evaluated by Chisquare statistics and by Phi, Cramer’s V and Contingency
coefficients. Statistical significance was presumed at p  0.05. Major trends within each
group were further examined by the Chisquared Automatic Interaction Detection
(CHAID) method[292], using the likelihood ratio Chi-square statistic as growing criteria
along with a Bonferroni 0.05 adjustment of probabilities. All analyses were carried out
with the SPSS, Statistical Package for Social Sciences (SPSS, Version 16, Chicago, IL).
50
STUDY 3
Thirty-one (n = 31) New Zealand white female rabbits (3-4 kg) were implanted
in an approved institutional animal care protocol with 1 textured tissue expander (nonfilled with 20 ml, Allergan, Santa Barbara, CA) and 2 textured breast implants (90 ml,
Allergan, Santa Barbara, CA). Prior to surgery the rabbit skin was washed with
Betadine® Surgical Scrub contains 7.5% povidone-iodine, followed by Betadine®
Solution containing 10% povidone-iodine (Purdue Products, Stamford, USA). The
surgical procedure was performed in an animal operating theatre following aseptic rules.
Penicillin G 40.000 u/Kg IM was administered just intraoperatively. Talc-free gloves
were used at all times during the procedure. Pockets were developed in the subpanniculus carnosis along the back region, with atraumatic dissection. Particular
attention was given to hemostasis, under direct vision avoiding blunt instrumentation
and there was no obvious bleeding. A sterile Op-site dressing was placed over the skin
around the incision before the tissue expander and the implants insertion to eliminate
contact with the skin[295]. A new pair of talc-free gloves was used to perform the tissue
expander and the implants insertion.
The rabbits groups were: 1) untreated implants and expander (Control; n = 10);
2) implants sprayed each one with 2 ml of fibrin (Tisseel/Tissucol®; Baxter Healthcare
Corporation, Vienna, Austria, Europe) and expander sprayed with 0.5 ml of fibrin and
(Fibrin; n = 11); 3) implants each one inoculated with 100 microlitres of a suspension
of coagulase-negative Staphylococci (108 CFU/ml - 0.5 density in McFarland scale) and
expander with 2.5 x 107 CFU/ml (CoNS; n = 10).
Rabbits were sacrificed at 4 weeks. Prior to sacrifice, each animal was
anesthetized and the dorsal back area shaved. A pressure measure device (Stryker®
instruments, Michigan, USA) was connected to the tissue expander port and
51
intracapsular pressures were recorded at each 5 ml increments previously to any incision
in the capsule. All capsule samples were submitted for histological and microbiological
evaluation. All implants and expander devices were also submitted for microbiological
evaluation.
Microbiological Assessments
As performed in STUDY 2.

Air : air samples (n = 36)
 Rabbit skin: 93 contact plates (31 brain-heart agar, 31 mannitol salt agar and 31
Sabouraud agar contact plates)
Histological Assessment
As performed in STUDY 2.
Statistical analysis
Data were grouped according to the type of product applied to the breast
implants, namely none (Control; n = 20), Tisseel/Tissucol® (Fibrin; n = 22) and
coagulase-negative Staphylococci (CoNS; n = 20). One-way analyze of variance either
parametric or nonparametric (Kruskal-Wallis H test) were performed to determine if
continuous variables (intracapsular pressure and histological measured thickness) were
equal, followed by post-hoc range tests to identify homogeneous subsets across groups.
Two-tailed independent pair t-tests and the nonparametric alternative Mann-Whitney U
tests were used. Categorical variables were evaluated by Chisquare statistics and by
Phi, Cramer’s V and Contingency coefficients. Statistical significance was presumed at
p  0.05. Major trends within each group were further examined by the Chisquared
52
Automatic Interaction Detection (CHAID) method
[292]
, using the likelihood ratio Chi-
square statistic as growing criteria along with a Bonferroni 0.05 adjustment of
probabilities. All analyses were carried out with the Statistical Package for Social
Sciences (SPSS, Version 16, Chicago, IL).
STUDY 4
Eleven (n = 11) New Zealand white female rabbits (3-4 kg) were implanted
according an approved institutional animal care protocol; each rabbit received 3
different textured breast implants (90 ml, Allergan, Santa Barbara, CA). Prior to surgery
the rabbit skin was washed with Betadine® Surgical Scrub containing 7.5% povidoneiodine, followed by Betadine® Solution containing 10% povidone-iodine (Purdue
Products, Stamford, USA). The surgical procedure was performed in an animal
operating theatre following aseptic rules. Penicillin G 40.000 u/Kg IM was administered
just intraoperatively. Talc-free gloves were used at all times during the procedure.
Pockets were developed in the sub-panniculus carnosis along the back region, with
atraumatic dissection. Particular attention was given to hemostasis, under direct vision
avoiding blunt instrumentation and there was no obvious bleeding. A sterile Op-site
dressing was placed over the skin around the incision before the implants insertion to
eliminate contact with the skin[295]. A new pair of talc-free gloves was used to perform
the implants insertion.
Each implant was placed beneath panniculus carnosis along the back (Figure
1A). The implant groups were: an untreated implant (Control); an implant impregnated
with COS (MW 1.4 kDa, Nicechem, Shanghai, China); and an implant impregnated
with LMWC (MW 107 kDa, Sigma-Aldrich, Sintra, Portugal). Both chitosan mixtures
possessed deacetylation degree in the range of 80−85%. Implants were prepared by
53
immersion in either COS (20.0 mg/mL) or LMWC (10.0 mg/mL) solutions with pH
adjusted to 5.8-5.9 for 2 hours. Implants were incubated at 37ºC in a flow chamber for 2
days, packed and sterilized by ethylene oxide.
Rabbits were sacrificed at 4 weeks. Prior to sacrifice, each animal was
anesthetized and a 5 mm incision was made directly over the implant through skin,
panniculus carnosis and capsule. A 100,000 molecular weight cutoff microdialysis
probe (CMA Microdialysis, Stockholm, Sweden) was placed by the capsule implant
interface and microdialysates were collected using sterile, normal saline solution (6
µl/min) for 1 hour.
Whole blood was obtained by venipuncture and serum was
collected after centrifugation (2000 gmin-1, 40C). Capsule samples were submitted to
histological and microbiological evaluations.
Microbiological Assessments
As performed in STUDIES 2 and 3.

Air : air samples (n = 20)

Rabbit skin: 33 contact plates (11 brain-heart agar, 11 mannitol salt agar and 11
Sabouraud agar contact plates)
Histological Assessment
As performed in STUDIES 2 and 3.
Microdialysis Assessment
The Invitrogen® Hu TNF-α US kit (Invitrogen®, Hu TNF-α Cat# KHC3014:1)
is a solid phase sandwich Enzyme-Linked-Immuno-Sorbent-Assay (ELISA). An
antibody specific for Hu TNF-α has been coated into wells of the microtiter strips
54
provided. The microdialysis fluid was pipetted into wells. During the first incubation,
the Hu TNF-α antigen binds to the immobilized (capture) antibody on one site, and to
the solution phase biotinylated antibody on a second site. After removal of excess
second antibody, Strepavidin-Peroxidase (enzyme) was added which binds to the
biotinylated antibody to complete the four-member sandwich. After a second incubation
and washing to remove the unbound enzyme, a substrate solution was added, which was
acted upon by the bound enzyme to produce color. The intensity of this colored product
was directly proportional to the concentration of Hu TNF-α presented in the original
specimen.
The protocol was repeated with the BioSource® Hu IL-8 US kit (BioSource®,
Hu IL-8 Cat# KHC0083/KHC0084).
Statistical analysis
Data were grouped according to the type of product applied to the implant,
namely none (Control), chitooligosaccharide mixture (COS) and low-molecular-weightchitosan (Chitosan) and analyzed separately for the 11 sacrificed rabbits at 4 weeks after
surgery. Two-tailed paired t-test and the nonparametric alternative Wilcoxon signed
rank test were used to determine whether continuous variables (histologic measured
thickness and dialysate levels of IL-8 and TNF-α) were likely to show differences
between Control and experimental groups. Categorical variables were evaluated by
Chisquare statistics and by Phi, Cramer’s V and Contingency coefficients. Statistical
significance was presumed at p  0.05. Major trends within each group were further
examined by the Chisquared Automatic Interaction Detection (CHAID) method [292],
using the likelihood ratio Chi-square statistic as growing criteria along with a
55
Bonferroni 0.05 adjustment of probabilities. All analyses were carried out with the
SPSS, Statistical Package for Social Sciences (SPSS, Version 17, Chicago, IL).
STUDY 5
Nineteen (n = 19) New Zealand white female rabbits were implanted in an
approved institutional animal care protocol with 1 textured tissue expander (non-filled
with 20 ml, Allergan, Inc., Santa Barbara, CA) and 2 textured breast implants (90 ml,
Allergan, Inc., Santa Barbara, CA). Prior to surgery, rabbit skin was washed with
Betadine® Surgical Scrub contains 7.5% povidone-iodine, followed by Betadine®
Solution containing 10% povidone-iodine (Purdue Pharma LP, Stamford, Connecticut).
The surgical procedure was performed in an animal operating theatre following aseptic
rules. Penicillin G 40.000 U/Kg was administered intramuscularly was administered just
intraoperatively. Talk-free gloves were used at all times during the procedure. Two 5
cm incisions and one 2,5 cm incision were made directly over the skin and subpanniculus carnosus to introduce the implants and the expander, respectively. Pockets
were developed in the sub-panniculus carnosis along the back region, with atraumatic
dissection. Particular attention was paid to hemostasis, under direct vision avoiding
blunt instrumentation and there was no obvious bleeding. A sterile Op-site dressing was
placed over the skin around the incision before the tissue expander and the implants
insertion to eliminate contact with the skin. A new pair of talc-free gloves was used to
perform the tissue expander and the implants insertion. Then, the implants and the tissue
expander with intact connecting tube and port were introduced. In the experimental
group, the introduction of triamcinolone-acetonide (Trigon® depot; Bristol-Myers
Squibb) into the implant and expander pocket was performed. All wounds were closed
with two planes of interrupted suture.
56
The rabbits groups were: 1) untreated implants and expander (Control; n = 10);
2) introduction of 1 ml (40 mg) of triamcinolone-acetonide into each implant pocket and
0.25 ml (10 mg) of triamcinolone-acetonide into each expander pocket (Triamcinolone;
n = 9). No fluid suction was performed in order to retain the prevent dilution of the
triamcinolone-acetonide (Trigon® depot; Bristol-Myers Squibb) in the surgical pocket.
Rabbits were sacrificed at 4 weeks. Prior to sacrifice, each animal was
anesthetized and the dorsal back area shaved. A pressure measure device (Stryker
Instruments, Kalamazoo, Michigan) was connected to the tissue expander port and
intracapsular pressures were recorded at each 5 ml increments previously to any incision
in the capsule. Then, a 5 mm incision was made directly over the implant through skin,
panniculus carnosus and capsule. A 100,000 molecular weight cutoff microdialysis
probe (CMA Microdialysis, Stockholm, Sweden) was placed by the capsule implant
interface and microdialysates were collected using sterile, normal saline solution (6
µl/min) for 1 hour.
Whole blood was obtained by venipuncture and serum was
collected after centrifugation (2000 gmin-1, 40C). All capsule samples were submitted
for histological and microbiological evaluation. All implants and expander devices were
also submitted for microbiological evaluation.
Microbiological Assessments
As performed in STUDIES 2, 3 and 4.

Air : air samples (n = 24)
 Rabbit skin: 57 contact plates (19 brain-heart agar, 19 mannitol salt agar and 19
Sabouraud agar contact plates)
57
Histological Assessment
As performed in STUDIES 2, 3 and 4.
Microdialysis Assessment
As performed in STUDY 4.
Statistical analysis
Data were analyzed by groups: Control (n = 20) and Triamcinolone (n = 18).
One-way analysis of variance (parametric or nonparametric) was performed to check if
the several means of continuous variables (histologic measured thickness and dialysate
levels of IL-8 and TNF-α) were equal, followed by post-hoc range tests to identify
homogeneous subsets across groups. A two-tailed independent pair t-test and the
nonparametric alternative Mann-Whitney U test were used to determine whether such
continuous variables were likely to show differences between control and experimental
group. Categorical variables were evaluated by Chisquare statistics and by Phi,
Cramer’s V and Contingency coefficients. Statistical significance was presumed at p 
0.05 and all analyses were carried out with the SPSS program.
58
4. Results
STUDY 1
Baseline descriptive information for the Cosmetic and Reconstructive patient
groups were presented in Tables IV and V, respectively.
Table IV. Baseline characteristics for the Cosmetic group.
Variable
No
%
69 (136)
Women with implants (breast implants)
Age at surgery in years, mean (range)
31.0 (1551)
Follow-up period in months, mean (range)
35.4 (1280)
Implant placement

Subpectoral
9
13.0

Subglandular
58
84.1

Dual plane Tebbets
2
2.9
Incision placement

Inferior periareolar
7
10.1

Axillary
21
30.4

Inframammary
41
59.5
Contraceptive drugs

No
30
43.5

Yes
39
56.5
59
Table V. Baseline characteristics for the Reconstructive group.
Variable
No
Women with implants (breast implants)
%
88 (115)
Age at surgery in years, mean (range)
48.6 (2573)
Follow-up period in months, mean (range)
48.5 (1296)
Symmetrizing breast

No
28
31.8

Breast implant (with or not mastopexy)
22
25

Breast reduction
33
37.5

Bilateral breast reconstruction
5
5.7
Hormone therapya

No
85
96.6

Yes
3
3.4
a
Including contraceptive drugs or hormone replacement therapy
Cosmetic patients were younger at the time of surgery when compared with
reconstructive patients (31.0 vs. 48.6 years). The average follow-up period was 35.6
months in the Cosmetic group when compared with 48.5 months in the Reconstructive
group.
Cosmetic patients reported contraceptive use (56.5%) while only 3.4% of
reconstructive patients reported contraceptive use or hormone replacement therapy.
Cosmetic patients also reported decrease use of psychotropic drugs (antidepressants,
antianxiety and hypnotics drugs) compared with reconstructive patients (23.2% .vs.
52.3%, respectively). One woman from each group (n = 2) had a connective tissue
disease (rheumatoid arthritis).
Among women in the Cosmetic group, the majority of silicone gel implants were
placed subglandularly (84.1%) and the surgical approach was through the
inframammary fold (58.0%). The majority of reconstructive patients had not received
radiotherapy (85.2%) or tamoxifen (67.1%); chemotherapy was administered in 51.1%;
60
the reconstructed breast was on the left side in 52.3% of the patients and 68.2% were
submitted to breast size symmetrization.
Clinical adverse events: acute
Acute complications were recorded in 20 reconstructive patients (8%) during the
follow-up period, with complications recorded as seroma (8.0%), hematoma (4.5%) and
perforation of the skin (3.2%).
61
Clinical adverse events: chronic
Chronic complication events were recorded and tabulated in Table VI.
Table VI. Chronic complications for both groups.
Chronic complications
Cosmetic group
(N = 69)
Reconstructive
group (N = 88)
No
%
No
%
Capsular contracture

No
57
82.6
46
52.3

Unilateral
9
13.0
41
46.5

Bilateral
3
4.4
1
1.2
Palpable implant folds

No
40
58.0
27
30.7

Unilateral
12
17.4
48
54.5

Bilateral
17
24.6
13
14.8
Visible skin wrinkles

No
59
85.5
72
81.8

Unilateral
7
10.1
14
15.9

Bilateral
3
4.4
2
2.3
Prolonged pain in the breast

No
59
85.5
78
88.7

Unilateral
4
5.8
9
10.2

Bilateral
6
8.7
1
1.1
Change of tactile sense

No
61
88.4
9
10.2

Unilateral
4
5.8
67
76.1

Bilateral
4
5.8
12
13.7
Overall, 81% (n = 127) of all women had 1 or more postoperative chronic
events, ranging from less severe effects (e.g.: change in tactile sense) to complications
requiring additional surgical interventions, such as severe capsular contracture. The
62
distribution of chronic complication frequency among women was: a) 23% of the
patients had one complication; b) 31% of the patients had two complications; c) 27% of
the patients had three 3 or more complications. From a temporal view of the clinical
onset of chronic complications, 3% of the patients were diagnosed from 0-12 months
postoperatively; 31% of the patients were diagnosed from 13-24 months; and 72% of
the patients were diagnosed from 0-60 months.
The most frequent chronic adverse effect was palpable implant folds (47.8% of
all cases), occurring in 42.0% of women from the Cosmetic group and in 69.3% from
the Reconstructive group. Change of tactile sense also had a high incidence (41.0% of
all cases) with 89.8% reporting changes in the Reconstructive group, not due to
reconstruction but mastectomy. For this reason, capsular contracture was the second
most common chronic complication, occurring in 34.4% of all women and in 23.1% of
all implantations. Capsular contracture incidence rates were significantly different
between the Cosmetic group (17.4% of women or 11.0% of implantations) and the
Reconstructive group (47.7% of women or 37.4% of implantations; p < 0.05). Other
chronic complications occurred less frequently (< 10% of all patients).
Furthermore, the occurrence of postoperative complications had a marked
influence upon satisfaction index, e.g., women without contracture were 1.6 times more
likely to consider the outcome either good or very good compared to women with
capsular contracture (RR = 1.6; 95% CI, 1.2, 2.2).
63
Capsular contracture characteristics
Baker capsular contracture grades for the cosmetic and reconstructive groups
were presented in Table VII.
Table VII. Capsular Contracture per implant for both groups.
Grade
Cosmetic group
Reconstructive group
I
121 (89.0%)
72 (62.6%)
II
5 (3.7%)
9 (7.8%)
III
2 (1.4%)
12 (10.4%)
IV
8 (5.9%)
22 (19.1%)
Total
136 (100%)
115 (100%)
As a percent of patients, Reconstructive group had 7.4 and 3.2 fold great
incidences of Baker III and IV grade capsular contractures compared to the Cosmetic
group. When examined as a function of clinical time when Baker grades were assigned,
44 women (76%) of the 58 total patients were diagnosed 2 years after surgery. In detail,
5 (7%) women from the Cosmetic group and 28 (32%) from the Reconstructive group
developed capsular contracture grade III/IV after the initial 2 years subsequent to
implantation. Overall, the rate of grade III/IV capsular contracture per woman during
the 8-year period of follow-up was 10.1% for patients undergoing cosmetic surgery, and
37.5% for breast reconstruction patients.
The occurrence of capsular contracture was associated with the duration of
follow-up period and age at time of surgery (Table VIII).
64
Table VIII. Identified variables related to capsular contracture for the entire group
(n = 157).
Capsular contracture (% of women)
Variable
No
Yes
Follow-up period
< 0.001

 42 months
40.1
10.8

> 42 months
25.5
23.6
Age at Surgery
< 0.001

 54 years
60.5
24.8

> 54 years
5.1
9.6
0.014
Hormone therapya

No
21.7
29.3

Yes
43.9
5.1
Type of group
< 0.001

Reconstructive
29.3
26.8

Cosmetic
36.3
7.6
a
p value
Including contraceptive drugs or hormone replacement therapy
Women with a follow-up period longer than 42-months (RR = 1.8; 95% CI, 1.3
to 2.4) or older women (RR = 3.6; 95% CI, 1.6 to 7.9 for an age of 54+ versus < 54
years) had increased incidences of capsular contracture (p < 0.001 for both
comparisons). Moreover, an increased capsular contracture was detected in the
Reconstruction group when compared to the Cosmetic group (RR = 1.7; 95% CI, 1.4 to
2.3; p < 0.001). No associations between capsular contracture cases and surgical
procedures or other personal characteristics were observed.
65
Using the CHAID decision tree (Figure 3), the type of group was identified as
the determining factor to develop capsular contracture. The first-level split produced
two initial branches: Cosmetic (no capsular contracture; percentage = 82.6%) and
Reconstructive (positive capsular contracture; percentage = 47.7%). The next splits
indicated the best predictor variables for the Reconstructive group, as the follow-up
period followed up by the age at surgery. Within that group, a follow-up period of 42
months or less was the best predictor for no capsular contracture (unadjusted percentage
= 74.3%) while a follow-up of more than 42 months was predictive for positive capsular
contracture (unadjusted percentage = 62.3%). For women with a follow-up of 42
months or less, capsular contracture was reported among 67.7% of women older than 54
years old compared to younger women (11.5%). The overall risk estimate according to
the classification tree was 0.240 (standard error of risk estimate 0.034), indicating that
75.8% of the cases will be classified correctly using the decision algorithm based upon
the current tree. The CHAID algorithm resulted in larger predictive values for
occurrence of capsular contracture (72.2%) than LR (57.4%).
66
Figure 3. Prediction tree of capsular contracture by Chi-squared Automatic Interaction
Detection algorithm.
CAPSULAR CONTRACTURE
NO 103 (65.6%)
YES 54 (34.4%)
TYPE OF GROUP p < 0.045
COSMETIC GROUP
NO 57 (82.6%)
YES 12 (17.4)
RECONSTRUCTIVE GROUP
NO 46 (52.3%)
YES 42 (47.7%)
FOLLOW UP p < 0.036
FOLLOW UP ≤ 42 months
NO 26 (74.3%)
YES 9 (25.7%)
FOLLOW UP > 42 months
NO 20 (37.7%)
YES 33 (62.3%)
YES 9 25.7
AGE AT SURGERY p < 0.026
AGE AT SURGERY ≤ 54 years
NO 23 (88.5%)
YES 3 (11.5%)
AGE AT SURGERY > 54 years
NO 3 (33.3%)
YES 6 (66.7%)
A second CHAID decision tree analysis was performed with grade II subjects
placed in the no capsular contracture group – similar to other reports – versus grade III
and IV subjects. The first-level split produced two initial branches: Cosmetic (no
capsular contracture or grade II; percentage = 89.9%) and Reconstructive (capsular
contracture grade III or IV; percentage = 37.5%). The next split indicated the best
predictor variable for the Reconstructive group, as the follow-up period. Within that
group, a follow-up period of 64 months or less was the best predictor for no capsular
contracture or grade II (unadjusted percentage = 73.4%) while a follow-up of more than
64 months was predictive for capsular contracture grade III or IV (unadjusted
percentage = 66.7%). The overall risk estimate according to the classification tree was
0.255 (standard error of risk estimate 0.035), indicating that 79.6% of the cases will be
classified correctly using the decision algorithm based upon the current tree.
67
Exogenous hormone use was reported in 56.5% of cosmetic patients (n = 39)
with one subject in menopause that used hormone replacement therapy; of the
remaining 68 women, 38 used contraceptives (Figure 4). Only 3.4% of reconstructive
patients (n = 3) used hormone therapy. Seventy-three patients were in menopause with 2
subjects using hormone replacement therapy. Fifteen women were premenopausal with
one using contraceptives.
Figure 4. Graphic of patients with or without menopause per type of group.
68
Subjects who were premenopausal or postmenopausal using hormone therapy
replacement, were grouped and analyzed as “estrogen protected” (Figure 5).
Figure 5. Graphic of patients protected or not by estrogen per type of group.
To clarify the relationships between menopause or women protected by estrogen
with capsular contracture rates per type of group, 2 cross-tabulations were performed
(Tables IX and X). No associations between capsular contracture and menopause or
estrogen status were observed.
Table IX. Cross-tabulation between capsular contracture and menopause per type of
group.
Type of group
Menopause
Cosmetic
Menopause
Reconstructive
Menopause
Yes
No
Total
Yes
No
Total
69
Capsular
contracture
Yes
No
0
1
12
56
12
57
36
37
6
9
42
46
Total
1
68
69
73
15
88
Table X. Cross-tabulation between capsular contracture and being protected or not by
estrogen per type of group.
Protected by estrogena
Type of group
Cosmetic
Protected by
estrogen
Reconstructive
Protected by
estrogen
a
Yes
Total
Yes
No
Total
Capsular
contracture
Yes
No
12
57
12
57
35
36
7
10
42
46
Total
69
69
71
17
88
Including all women before menopause or in menopause with hormone replacement therapy.
STUDY 2
Intracapsular pressure
No significant differences were observed regarding the pressure-volume curves
between the Control and the experimental groups at baseline (tissue expander
introduction) or at 2 weeks. At 4 weeks, rupture of 6 capsules in the Control group, 5
capsules in the Blood group and 1 capsule in Thrombin group, during pressure
measurement were observed; no capsule ruptures in the Fibrin group were noted. To
avoid too less sampling, the ruptured capsules were not excluded from statistical
analyses but was stated that the pressure levels measured before capsule rupture were
maintained after further additional saline was added. At 4 weeks, significant decreased
intracapsular pressures were registered in the Fibrin (p  0.0006) and Thrombin (p 
0.003) groups (Figure 6).
70
Figure 6. The pressure-volume curves at 4 weeks; there was a significant difference in
intracapsular pressure in the Thrombin (FloSeal®) and Fibrin (Tissucol/Tisseel®)
experimental groups.
Mean pressure (mmHg)
.
100
90
80
70
60
50
40
30
20
10
0
Control
Blood
Thrombin
Fibrin
0
10
20
Additional ml saline
30
Histology
The average capsular thicknesses were similar among all groups at 2 and 4
weeks (Table XI). At 2 weeks, mixed type of inflammatory cells was predominantly
observed in rabbit capsules and no statistically differences were found (Table XII). At 4
weeks, mononuclear type of inflammatory cells was predominant in the Control, Blood
and Thrombin groups; in the Fibrin group mixed type of inflammatory cells was
predominant but no statistically significant differences were observed (Table XII). Both
at 2 and 4 weeks trends of intensity of inflammation showed no significant difference
(Table XIII).
71
Table XI. Average capsular thickness of Control versus experimental groups.
Group
2 weeks (mm)
4 weeks (mm)
Control
0.83 ± 0.085
0.64 ± 0.078
Blood
1.02 ± 0.207
0.78 ± 0.572
Fibrin
0.89 ± 0.082
0.72 ± 0.083
0.90 ± 0.064
0.71 ± 0.105
(Tissucol/Tisseel®)
Thrombin
(FloSeal®)
Table XII. Outcomes for type of inflammatory cells of Control versus experimental
groups.
Group
Type of inflammatory cells
2 weeks (%)
4 weeks (%)
Control
Mononuclear
22.2
55.6
Polymorph
0
0
Mixed
77.8
44.4
Mononuclear
33.3
55.6
Polymorph
0
0
Mixed
66.7
44.4
Mononuclear
11.1
22.2
Polymorph
0
0
Mixed
88.9
77.8
Thrombin
Mononuclear
22.2
77.8
(FloSeal®)
Polymorph
0
0
Mixed
77.8
22.2
Blood
Fibrin
(Tissucol/Tisseel®)
72
Table XIII. Outcomes for intensity of inflammation of Control versus experimental
groups.
Group
Intensity
2 weeks (%)
4 weeks (%)
Control
Mild
11.1
55.6
Moderate
77.8
44.4
High
11.1
0
Mild
33.3
33.3
Moderate
66.7
66.7
High
0
0
Mild
0
22.2
Moderate
66.7
33.3
High
33.3
44.4
Thrombin
Mild
11.1
66.7
(FloSeal®)
Moderate
77.8
33.3
High
11.1
0
Blood
Fibrin
(Tissucol/Tisseel®)
Fibrosis was developed in all capsules at 2 and 4 weeks and no significant
differences were observed regarding the organization of the collagen fibers between the
Control and the experimental groups. At 2 weeks, dense >25% connective tissue in the
Control group and loose or dense 25% connective tissue in the Blood group were
observed (p = 0.023). Both at 2 and 4 weeks, increased angiogenesis was observed in
the Control group (moderate or high) versus the Blood group (negative or mild) (p =
0.018). At 4 weeks, significant differences in the fusiform cells density were observed
between the Control and the Blood groups (p = 0.047), with mild in the Control group
and moderate in the Blood group.
73
Microbiology
Bacteria were isolated in 53% (38 of 72) of the capsules at 2 and 4 weeks, and in
47% (34 of 72) of tissue expanders. The isolates included: coagulase-negative
Staphylococci (41%), Escherichia coli (10%), Staphylococcus aureus (8%),
Pseudomonas spp. (0.7%), and other gram-negative bacilli (0.7%). In capsules, the
predominant isolates were coagulase-negative Staphylococci detected in 53% (19 of 36)
at 2 weeks and in 33% (12 of 36) at 4 weeks. In tissue expanders, coagulase-negative
Staphylococci were found in 44% (16 of 36) at 2 weeks and in 22% (8 of 36) at 4
weeks. Capsules yielded a single isolate in 43% (31) of cases and more than one in 10%
(7) of cases; tissue expanders yielded a single isolate in 32% (23) of cases and more
than one in 15% (15) of cases. No fungi were recovered from the removed capsules or
tissue expanders of all rabbits.
Similar bacterial isolates were cultured from the rabbit’s skin. The predominant
isolates were coagulase-negative Staphylococci, found in 16 of all 18 sacrificed rabbits
(89%). Bacterial isolates from rabbit’s skin were similar to those found in capsules and
tissue expanders. Coagulase-negative Staphylococci were also isolated from all air
samples. Other common airborne isolates included gram-positive bacilli and
Staphylococcus aureus; less frequently Penicillium spp., Aspergillus niger and
zygomicetes were recovered from the operation room air.
Statistical analyses revealed no significant differences in the frequency of
culture positivity and the type of bacterial isolates among all the groups; also, no
significant correlation between the microbiological and the histological data were
found.
74
CHAID modeling associations
At 4 weeks statistical analysis with CHAID modeling showed association of
intracapsular pressure measured at 20 ml, for the Control and the Fibrin groups. The
determining factor for intracapsular pressure at 4 weeks was the type of inflammatory
cells (Figure 7 a/b). The CHAID analysis showed in both trees that mixed type of
inflammatory cells was correlated with decreased intracapsular pressure and
mononuclear type of inflammatory cells was correlated with increased intracapsular
pressure. In the Control tree, in the capsules with mononuclear type of inflammatory
cells, the moderate inflammation was correlated with decreased pressure while capsules
with mild inflammation had increased pressure.
CHAID classification analyses using intracapsular pressure measured at 20 ml,
for the Fibrin and the Thrombin groups showed that the determining factor for
intracapsular pressure at 4 weeks was the kind of bacteria isolated from tissue expanders
(Figure 8 a/b). Escherichia coli, Pseudomonas spp. and negative cultures (other than
Staphylococcus and no contaminated) were correlated with decreased intracapsular
pressures.
Coagulase-negative
Staphylococci
and
Staphylococcus
(Staphylococcus) were correlated with increased intracapsular pressures.
75
aureus
Figure 7. Decision tree by CHAID algorithm for histological data at 4 weeks. (a)
Control group; (b) experimental Fibrin (Tissucol/Tisseel®) group.
PRESSURE
≤ 81: 5 (55.6%)
>81: 4 (44.4%)
TYPE OF INFLAMMATORY CELLS p < 0.0007
MIXED
≤ 81: 4 (100%)
>81: 0 (0%)
MONONUCLEAR
≤ 81: 1 (20%)
>81: 4 (80%)
INTENSITY p < 0.025
MILD
≤ 81: 0 (0%)
>81: 4 (100%)
MODERATE
≤ 81: 1 (100%)
>81: 0 (0%)
Figure 7 – (a)
PRESSURE
< 70: 6 (66.7%)
≥70: 3 (33.3%)
TYPE OF INFLAMMATORY CELLS p < 0.017
MIXED
< 70: 6 (85.7%)
≥70: 1 (14.3%)
MONONUCLEAR
< 70: 0 (0%)
≥70: 2 (100%)
Figure 5 – (b)
Figure 7 – (b)
76
Figure 8. Classification tree by CHAID algorithm for microbiological data at 4 weeks.
(a) experimental Fibrin (Tissucol/Tisseel®) group; (b) experimental Thrombin
(FloSeal®) group. NO: other than Staphylococci and no contaminated includes
Escherichia coli, Pseudomonas spp. and negative cultures; S: Staphylococci includes
coagulase-negative Staphylococci and Staphylococcus aureus.
PRESSURE
< 70: 6 (66.7%)
≥70: 3 (33.3%)
MICROBIOLOGY p < 0.001
NO (no staphylococci)
< 70: 6 (100%)
≥70: 0 (0%)
S (Staphylococci)
< 70: 0 (0%)
≥70: 3 (100%)
Figure 8 – (a)
PRESSURE
< 64: 5 (55.6%)
≥64: 4 (44.4%)
MICROBIOLOGY p < 0.001
NO (no Staphylococci)
< 64: 5 (83.3%)
≥64: 1 (16.7%)
S (Staphylococci)
< 64: 0 (0%)
≥64: 3 (100%)
Figure 8 – (b)
77
STUDY 3
Statistical analyses revealed no significant differences in histological and
microbiological results between breast implants and tissue expanders (data not shown).
Intracapsular pressure
During pressure measurements, 5 (50%) capsules ruptured in the Control group
and 5 (50%) capsules ruptured in the CoNS group. To avoid too less sampling, the
ruptured capsules were not excluded from statistical analyses but, in such cases, the
pressure value measured before rupturing was maintained after further additional ml
saline added. Significant decreased intracapsular pressures were registered for the Fibrin
group compared with the Control and the CoNS groups (p  0.001; p  0.05) (Figure 9).
Statistical analyses revealed no significant differences between the CoNS and the
Control groups (Figure 9).
Figure 9. The pressure-volume curves; there was a significant difference in
intracapsular pressure in the Fibrin (Tissucol/Tisseel®) experimental group.
78
Histology
Average capsular thicknesses were 0.81 ± 0.21 mm, 0.47 ± 0.13 mm and 1.06 ±
0.29 mm in the Control, Fibrin and CoNS groups. Capsular thickness was not
statistically homogeneous across the 3 groups (p  0.001). Then, three subsets of similar
means were found out by applying pos-hoc range tests, namely a first one comprising
the Fibrin group (with the thinnest capsule), a second comprising the Control group, and
a third one with the CoNS group (with the thickest capsule).
CHAID statistical modeling showed correlation between intracapsular pressure
measured at 20 ml and thickness for the Control and the Fibrin groups (Figure 10 a/b);
decreased intracapsular pressure was associated with thinner capsule for both groups,
and the opposite was also true.
Figure 10. Decision tree by CHAID algorithm for thickness. (a) Control group; (b)
experimental Fibrin (Tissucol/Tisseel®) group.
PRESSURE
≤ 173: 12 (60%)
>173: 8 (40%)
THICNESS p < 0.002
≤0.8 mm
≤ 173: 12 (80%)
>173: 3 (20%)
>0.8 mm
≤ 173: 0 (0%)
>173: 5 (100%)
Figure 10- (a)
79
PRESSURE
≥140: 14 (63.6%)
<140: 8 (36.4%)
THICNESS p < 0.004
≤0.5 mm
≥140: 12 (85.7%)
<140: 2 (14.3%)
>0.5 mm
≥140: 2 (25%)
<140: 6 (75%)
Figure 10- (b)
A mixed type of inflammatory cells was the most common finding in the Control
and the Fibrin groups, but in the CoNS group, the polymorph type became predominant
(Table XIV). Significant differences were observed between the Control and the CoNS
groups (CoNS: p = 0.0001), between the CoNS and the Fibrin groups (p = 0.0009), but
not between the Control and the Fibrin groups.
Intensity of inflammation was moderate in the Control and the Fibrin groups and
mild in the CoNS group (Table XIV). Significant differences were found between the
Control and the CoNS groups (p = 0.011), between the CoNS and the Fibrin groups (p =
0.0058) but not between the Control and the Fibrin groups. Significant correlations
between the intensity of inflammation and the type of inflammatory cells for the Control
(p = 0.005) and the Fibrin (p = 0.006) groups were observed.
80
Table XIV. Outcomes for capsule inflammation of Control versus experimental groups.
Group
(%)
Intensity
(%)
Mononuclear
25.0
Mild
30.0
Polymorph
0
Moderate
70.0
Mixed
75.0
High
0
Mononuclear
13.6
Mild
31.8
Polymorph
13.6
Moderate
59.1
Mixed
72.8
High
9.1
Mononuclear
35.0
Mild
70.0
Polymorph
50.0
Moderate
30.0
Mixed
15.0
High
0
Type of inflammatory
cells
Control
Fibrin
(Tissucol/Tisseel®)
CoNS
Fibrosis was detected in all capsules; no significant differences regarding the
fusiform cells density among all groups were observed. Significant differences in the
connective tissue were found between the Control and the Fibrin groups (p = 0.005),
and between the CoNS and the Fibrin groups (p = 0.0007), with dense >25% connective
tissue in the Control and the CoNS groups and loose or dense 25% connective tissue in
the Fibrin group.
Significant differences in the organization of the collagen fibers were observed
between the Control and the Fibrin groups (p = 0.019), and between the CoNS and the
Fibrin groups (p = 0.0039), with haphazard collagen fibers in the Control and the CoNS
groups and fibers arrayed parallel in the Fibrin group.
Significant differences in the angiogenesis were found between the Control and
the Fibrin groups (p = 0.003), and between the CoNS and the Fibrin groups (p = 0.016),
81
with moderate or high in the Control and the CoNS groups and negative or mild in the
Fibrin group.
Microbiology
Bacteria were isolated in 31% (19 of 62) of removed capsules, and in 84% (56 of
62) of the removed implants (Table XV). The predominant isolates were coagulasenegative Staphylococci, which were found in 16% (10 of 62) of all culture positive
capsules, and in 60% (37 of 62) of culture positive implants. Overall, 97% and 90% of,
respectively, culture positive capsules and implants yielded a single isolate, while 3%
and 10% yielded two. No bacteria were detected on 69% of the removed capsules and
on 16% of the removed implants. No fungi were recovered from the removed capsules
or implants among all groups.
82
Table XV. Bacterial isolates from capsules and implants removed from the sacrificed
rabbitsa.
Bacteria
Groupb
Number of positive
cultures
Coagulase-negative Staphylococci
Staphylococcus aureus
Gram-positive bacilli
Micrococcus spp.
Capsules
Implants
Control
2 (10%)
13 (65%)
Fibrin
2 (9%)
9 (41%)
CoNS
6 (30%)
15 (75%)
Control
2 (10%)
2 (10%)
Fibrin
1 (5%)
7 (32%)
CoNS
0 (0%)
2 (10%)
Control
1 (15%)
1 (5%)
Fibrin
3 (14%)
4 (18%)
CoNS
0 (0%)
2 (10%)
Control
0 (0%)
0 (0%)
Fibrin
0 (0%)
0 (0%)
CoNS
2 (10%)
1 (5%)
a
62 capsules and 62 implants were obtained from 31 rabbits
Data collected from groups Control (10 rabbits; 20 capsules and 20 implants), Fibrin
(11 rabbits; 22 capsules and 22 implants) and CoNS (10 rabbits; 20 capsules and 20
implants)
b
Statistical analysis revealed no significant differences in the type of bacteria and
in the frequency of culture positivity among the study groups. Also, there was no
significant association between microbiological and histological data.
Similar bacteria were isolated from the rabbit’s skin. The predominant isolates
were coagulase-negative Staphylococci, which was found in 37 of all 45 sacrificed
rabbits (82%), followed by gram-positive bacilli (60%), Staphylococcus aureus (33%)
83
and Micrococcus spp. (9%). Other isolates found were Enterococcus hermanii, S.
harmolyticum and Proteus mirabilis, though much less frequently. No skin sample was
culture-negative while thirty-five samples yielded more than one isolate. The bacterial
isolates from rabbit’s skin were similar to those from the removed capsules and
implants. Finally, coagulase-negative Staphylococci were also cultured from all the air
samples; other airborne isolates were gram-positive and negative bacilli, such as
Micrococcus spp., Cryptococcus laurentii, Acinetobacter lwofii and Enterococcus
agglomurans. Fungal species, such as Penicillium spp., Aspergillus niger, A. flavus and
A. fumigatus were recovered from the operation room air, Penicillium being the most
common fungal isolate one.
In the CoNS group one animal developed a clinical contracture Baker grade IV,
in one breast implant (Figure 11). The capsular thickness measured 1.70 mm and was
the largest one among all capsules. The type of inflammatory cells was polymorph with
moderate intensity. Histological evaluation of fibrosis revealed dense 25-50%
connective tissue, haphazard collagen fibers, moderate fusiform cells density and
moderate angiogenesis. Capsule and breast implant, were both infected with
Micrococcus spp.; no other bacteria or fungi were detected.
84
Figure 11. Rabbit 31 from the CoNS group with a clinical contracture grade IV in the B
implant.
STUDY 4
Clinical
In the Control group, 1 of the 11 implants was ulcerated and none had developed
clinical capsular contracture. In the COS group, 3 of the 11 implants were ulcerated and
no implant had an observation of clinical capsular contracture. The Chitosan group had
1 ulcerated implant and all 11 implants had grade III/IV capsular contracture (Figure
12B). All Chitosan group capsules were extremely thick, opaque, stiff and resistant to
cutting (Figure 12C); the implants were constricted and surface folding was observed.
85
Figure 12.
A) Rabbit with control implant, COS implant and Chitosan implant (contracture
grade IV)
B) Chitosan implant- contracture grade IV
C) Chitosan implant - extremely thick, dense and opacity capsule
D) Chitosan implant - hematoxylin-eosin stain magnification 100x with apoptotic
cells (cells have hiperchromatic and fragmented nuclei)
Histology
The average capsular thickness was 0.418 ± 0.160 mm in the Control group,
0.6364 ± 0.216 mm in the COS group, and 2.746 ± 0.817 mm in the Chitosan group.
Capsular thicknesses were found to be statistically different among the 3 groups;
capsular thicknesses from the Control group, were different from the COS group (p =
86
0.035) and from the Chitosan group (p= 0.003); capsular thicknesses were different
between COS and Chitosan groups (p = 0.003).
No significant differences were observed regarding the type of inflammatory
cells and intensity of capsule inflammation among the groups (Table XVI).
Table XVI. Outcomes for type and intensity of inflammatory cells of Control versus
experimental groups.
Groups
Type of inflammatory cells
(%)
Intensity
(%)
Control
Mononuclear
Polymorph
Mixed
9.1
36.4
54.5
Mild
Moderate
High
72.7
27.3
0.0
COS
Mononuclear
Polymorph
Mixed
9.1
27.3
63.6
Mild
Moderate
High
54.5
45.5
0.0
Chitosan
Mononuclear
Polymorph
Mixed
0.0
45.5
54.5
Mild
Moderate
High
36.4
63.6
0.0
Apoptotic cells and necrosis (Figure 12D) were observed strongly in Chitosan
group. Fibrosis was a component of all capsules and no significant difference was
found regarding the organization of the collagen fibers (mainly arrayed in parallel in all
groups), fusiform cells density and angiogenesis among all the groups. Regarding the
characteristics of connective tissue (either loose or dense), significant differences were
found between the Control and the Chitosan groups (p = 0.001); Control group had
loose or dense 25% connective tissue and Chitosan group dense >25% connective
tissue (mainly dense 25-50%).
87
Microbiology
Bacteria were isolated from 36.4% (12 of 33) capsules, and from 78.8% (26 of
33) implants. The organisms cultured (Table XVII) included coagulase-negative
Staphylococci, Staphylococcus aureus, gram-negative bacilli and Enterococcus spp..
Among all the capsules that yielded bacteria, 11 of 12 capsules harboured coagulasenegative Staphylococci (91.7%) and Enterococci were associated with 1 capsule (8.3%).
The same trend was observed in excised implants. In 20 of 26 implants that yielded
bacteria, coagulase-negative Staphylococci were cultured from 76.9% and Enterococcus
spp. was associated with 1 capsule (3.8%). In contrast to capsules, 4 of 26 bacterial
contaminated implants harboured gram-negative bacilli (15.4%) and 1 of 26
Staphylococcus aureus (3.8%).
Table XVII. Bacterial isolates from capsules and implants samples removed from
sacrificed rabbits.
Number of Positive Cultures
Capsules
Bacteria
Implants
Coagulase-negative
Staphylococci
Control
COS
Chitosan
4 (36.4%)
5 (45.5%)
2 (18.2%)
9 (81.8%)
8 (72.7%)
3 (27.3%)
Staphylococcus aureus
Control
COS
Chitosan
0 (0%)
0 (0%)
0 (0%)
0 (0%)
1 (9.1%)
0 (0%)
Gram-negative bacilli
Control
COS
Chitosan
0 (%)
0 (%)
0 (0%)
2 (18.2%)
1 (9.1%)
1 (9.1%)
Enterococcus spp.
Control
COS
Chitosan
0 (0%)
1 (9.1%)
0 (10%)
1 (9.1%)
0 (%)
0 (%)
88
Overall, 39.4% (13 of 33) and 63.6% (21 of 33) of respectively culture positive
capsules and implants yielded a single isolate, while 0% (0 of 33) and 9.1% (3 of 33)
yielded more than one. No fungi were recovered from either capsules or implants.
No significant differences in the frequency of culture positivity and type of
bacterial isolates were observed among all the study groups. No significant association
between microbiological and histological data were also observed.
Considering rabbit’s skin isolates, the predominant isolate was again coagulasenegative Staphylococci, which was formed in all rabbits. Bacterial isolates from skin
were similar to those from capsules and implants. Coagulase-negative Staphylococci
and gram-positive bacilli were isolated from all the air samples of the operation room,
along with Penicillium spp. and Aspergillus spp..
Immunology
Interstitial fluid of IL-8 levels decreased from 89.4 ± 26.7 mg/ml in the Control
group to 78.3 ± 32.7 mg/ml in the COS group, and to 66.8 ± 17.9 mg/ml in the Chitosan
group. Significant differences were observed in IL-8 levels between the Control and the
Chitosan groups (p = 0.028).
Levels of TNF-α decreased from 143.9 ± 123.8 mg/ml in the Control group to
96.8 ± 38.5 mg/ml in the COS group, and to 81.5 ± 31.8 mg/ml in the Chitosan group.
Statistical analysis revealed no significant differences in the dialysate levels of TNF-α
among all the groups. There was correlation between IL-8 and TNF-α in the Control
group (p < 0.001), but it was not found in the COS (p = 0.073) and the Chitosan groups
(p = 0.099).
89
STUDY 5
Statistical analyses revealed no significant differences in histological and
microbiological results between breast implants and tissue expanders (data not shown).
The expanders were included in the protocol to determine the pressure-volume curves.
Clinical
In the Triamcinolone group (Figure 13) the capsule was thinner and more
transparent than those of the Control group.
Figure 13. Capsule in the Triamcinolone experimental group.
90
Intracapsular pressure
During pressure measurements, 5 (50%) capsules ruptured in the Control group.
To avoid too less sampling, the ruptured capsules were not excluded from statistical
analyses but, in such cases, the pressure value measured before rupturing was
maintained after further additional mls saline added. Pressure-volume curves were
generated for all rabbits sacrificed. Statistical analyses revealed no significant
differences between the Triamcinolone and the Control groups (Figure 14).
Figure 14. The pressure-volume curves.
91
Histology
Significant decreased capsular thickness was registered for the Triamcinolone
group compared with the Control group (p  0.001) (Table XVIII).
A mixed type of cells was the most common finding in the Control and the type
mononuclear of cells was the most common finding in the Triamcinolone group (Table
XVIII). Significant differences were found between the Control group and the
Triamcinolone group ( p = 0.0003).
Regarding the intensity of inflammation, a significant difference was observed
between the Triamcinolone and Control groups (p =0.009), with mild in the
Triamcinolone group and moderate in the Control group (Table XVIII).
No significant differences regarding the fusiform cells density, connective tissue,
organization of the collagen fibers, between the Control and Triamcinolone groups were
observed. Significant differences were found in angiogenesis between the Control group
where it is basically moderate or high and the Triamcinolone (p = 0.007) group, where it
was negative or mild.
Table XVIII. Outcomes for capsular thickness and inflammation of Control versus
Triamcinolone Groups.
Group
Control
Triamcinolone
Capsular
Type of
thickness (mm)
inflammatory cells
0.81 ±
Mononuclear
0.209
Polymorph
(%)
25.0
0
Intensity
(%)
Mild
30.0
Moderate
70.0
Mixed
75.0
High
0
0.53 ±
Mononuclear
83.3
Mild
72.2
0.136
Polymorph
Moderate
27.8
Mixed
92
0
16.7
High
0
Microbiology
Statistical analysis revealed no significant difference in the type of bacteria and
in the frequency of culture positivity bacteria between the Control and Triamcinolone
groups regarding either implants or capsules. Also, there was no significant association
between microbial presence and histological data. The predominant isolate was
undoubtedly coagulase-negative staphylococci, which was identified predominantly in
the removed implants (Table XIX).
Isolated bacteria from rabbits’ skin and from the room air were statistically
similar to those from the removed capsules and implants, being coagulase-negative
staphylococci the prevailing one.
No fungi were recovered from the removed capsules or implants or skin samples
of all rabbits. Fungal species, such as Penicillium spp. and Aspergillus were recovered
from the operation room air.
Table XIX. Bacteria isolated from Capsule and Implant samples removed from all
sacrificed Rabbitsa.
Number of Positive Cultures
Bacteria
Coagulase-negative staphylococci
Group
Control
Triamcinolone
Staphylococcus Aureus
Control
Triamcinolone
Bacillus gram-positive
Control
Triamcinolone
a
Capsules
2 (10%)
6 (33%)
13 (65%)
14 (78%)
2 (10%)
2 (11%)
2 (10%)
2 (11%)
1 (15%)
2 (11%)
1 (5%)
2 (11%)
Data collected from groups Control (10 rabbits; 20 capsules and 20
implants),and Triamcinolone (9 rabbits; 18 capsules and 18 implants) .
93
Implants
Immunology
The dialisate levels of IL-8 decreased from 115.56 ± 128.03 mg/ml in the
Control group, to 54,41 ± 31.21 mg/ml in the Triamcinolone group. Statistical analysis
revealed no significant difference in the dialisate levels of IL-8 between the Control and
the Triamcinolone groups.
The dialisate levels of TNF-α decreased from 328.62 ± 307.55 mg/ml in the
Control group to 148.9177 ± 211.92273 mg/ml in the Triamcinolone group. Statistical
analysis revealed no significant difference in the dialisate levels of TNF-α between the
Control and the Triamcinolone group.
There is correlation between IL-8 and TNF- α in the Control group (p < 0.001)
and in the Triamcinolone group (p = 0.036)
94
5. Discussion
STUDY 1
Capsular contracture in a Portuguese population
In our report, the occurrence of local complications, the frequency, severity and
long-term
sequela
were
in
the
reported
range
as
described
in
other
studies[12],[13],[14],[15],[16],[17],[18],[19].
TableIII[12],[13],[14],[18],[19],[57]
,[85],[86],[87],[88],[89],[90],[91],[92],[93],[94]
demonstrates that
reported capsular contracture rates vary widely due to authors’ reporting various Baker
classification rates and follow-up time periods. These data showed incidence
complications were elevated in reconstruction patients compared to cosmetic
patients[95],[13]. No acute complications occurred in the Cosmetic group and all chronic
complications were less prevalent in this group. In our study, women with breast
implants due to cosmetic reasons had a lower body mass index than women with breast
reconstruction, similarly to previous study which compared breast augmentation with
breast reduction and general population[9].
In this study, 16% of capsular contracture (Baker III to IV) was diagnosed after
a 1.6-year period following initial breast implantation. There were no significant
associations between surgical route or implant placement, and any postoperative
complication. Like Henriksen et al.[58], no significant associations were observed
between body index mass, smoking habits, alcohol consumption, hormone therapy and
capsular contracture in our study groups.
95
Capsular contracture, type of cohort, Baker II subjects and follow-up period
Capsular
contracture
implantation[13],[14],[20],[58].
may
be
apparent
within
the
first
year
after
However, in our study, about 76% of cases of capsular
contracture (Baker II to IV) appeared just following 2 years; 10.1% and 37.5% of severe
capsular contracture (Baker III to IV) occurred in the Cosmetic and Reconstructive
groups, respectively, during the 8 years period of follow-up. Breiting et al.
[9]
reported
18% of severe breast pain, indicative of severe capsular contracture and, in a previous
study, involving a subgroup of this population they had diagnosed 45% of capsular
contracture (Baker II to IV) after a 5 years period following breast implantation[96].
Capsular contracture may also be symptomatic several years after surgery[9],[20],[58],[59].
Using the CHAID decision tree, the determining factor for capsular contracture
was the type of group; the next splits indicated the best predictor variables for the
Reconstructive group, as being the follow-up period; once considered no capsular
contracture versus capsular contracture the follow-up period should be longer than 3
years and 6 months. However, if considering no capsular contracture including grade II
subjects versus grade III or IV subjects, a longer follow up period of 5 years and 4
months was determined. It is interesting that both CHAID tree decision analyses had the
same qualitative splits but with longer follow up time periods in grade III or IV
subjects. This is expected as breast capsule formation is thought to develop from grade
II to grade III, and grade III to grade IV. These results underscore the importance of
considering grade II as an important clinical observation that should be included in the
capsular contracture analyses. Thus, we believe that a follow-up period longer than 42
months from grade II and reconstructive patients should be considered when studying
local complications among women receiving breast implants.
96
Estrogens, menopause and capsular contracture
It is well known the protective role of estrogens in the progression of liver
fibrosis[97],[98] and the fact that estrogen deprivation was being associated with declining
dermal collagen content and impaired wound healing[99], nevertheless there are no
reports concerned with menopause nor estrogens versus capsular contracture. The
authors for the first time report no association between capsular contracture and
menopause or estrogen status. Therefore, the pathophysiology of capsule formation and
subsequent contracture developing metabolic pathways are not estrogen derived.
Limitation and strength of the study
The main limitation of this study was the relatively small sample size and thus
limited statistical power to observe relationships with rare outcomes, especially in the
Cosmetic group.
One strength of this study was the statistical analyses of these data among the 2
groups using the CHAID method, a sophisticated algorithm used in many other
disciplines, adjusting the probability of a single variable among multiple variables.
STUDY 2
The major findings of our study were observed on capsules and tissue expanders
in the rabbits sacrificed at 4 weeks. Compared to the Control group, in the Fibrin and
the Thrombin groups significantly decreased intracapsular pressures were measured.
The Fibrin group was the only group without capsule ruptures during the pressure
measurement. For both the Control and the Fibrin groups, mixed type of inflammatory
cells was correlated with decreased intracapsular pressures while mononuclear type of
inflammatory cells was correlated with increased intracapsular pressures. For both the
97
Fibrin and the Thrombin groups, other bacteria than Staphylococci or negative cultures
were correlated with decreased intracapsular pressures and Staphylococci were
correlated with increased intracapsular pressures. In the Blood group increased fusiform
cells density were observed compared to the Control group. Increased angiogenesis was
observed in the Control group compared to the Blood group. Average capsular
thicknesses, type and intensity of the inflammatory cells, connective tissue and
organization of the collagen fibers were similar among all groups. Also the bacterial
isolates from capsules, tissue expanders and rabbit skin were similar among the four
groups. In capsules, tissue expanders and rabbit skin the predominant isolates were
coagulase-negative Staphylococci, which were also isolated from all air samples. No
fungi were recovered from capsules, tissue expanders or rabbit’s skin, although being
isolated from all air samples.
It should be addressed that this study was performed with tissue expanders to
measure the capsule pressure directly, o achieve more accurate results[144]. Similar
capsules and increased pressure levels were observed in both the Control and Blood
groups. Based on wound healing principles we may conclude that increased pressure
levels and capsule rupture rates were correlated with contracture[145]. The fact that
increased angiogenesis is related with fibrosis was demonstrated, supporting the major
trends observed in the capsule contracture development in the Control group of this
study[129],[144],[296].
FloSeal® requires blood for activity; with the Thrombin group results we may
conclude that an active hemostasis is indispensable to prevent capsular contracture,
although unnecessary with a hemostatic commercial product.
On the other hand, the Fibrin group had mixed type of inflammatory cells
correlated with decreased intracapsular pressures compared with the Control group,
98
which is consistent with other reports observing that the activation of fibrosis in the
early implant period may be the major mechanism for capsular contracture
development[125]. In our study, type of inflammatory cells was not significantly
correlated with capsular thickness which is consistent with Siggelkow et al.[125] results.
Sead et al.[249] studied fibrin sealant prepared from Tisseel kit without aprotinin and
observed the ability to reduce extracellular matrix and TGF-β1, especially from
adhesion fibroblasts, which may indicate a role in reduction of postoperative adhesion
development. It is well known that fibrosis is associated with excessive collagen
extracellular matrix (ECM) formation and cells proliferation and activation of
myofibroblasts. In this context, macrophages and mast cells have been implicated as
important participants in the inflammatory process involving fibrosis[124]. Macrophages
contribute to this process by the production of TGF-β1 and IL-6[162]. In the study by
Ruiz-de-Erenchun et al.[297], TGF-β1 inhibitor peptide applied in a matrix with
tetraglycerol dipalmitate was significantly effective in achieving a reduction in
periprosthetic fibrosis after placement of silicone implants. Interestingly, in the Fibrin
group, mixed type of inflammatory cells was correlated with decreased intracapsular
pressures, however if infected by Staphylococcus the intracapsular pressures increased.
Our results suggest the role of fibrin in preventing capsular contracture; and that the
bacterial colonization of mammary implants may be partially responsible for capsule
contracture, and coagulase-negative Staphylococci may play a relevant role
[71],[72],[130],[131],[132],[133],[134],[135],[136],[137],[138],[139],[140],[141],[142]
. It is reported in literature
that infection of implanted medical devices was commonly mediated by formation of
bacterial biofilms[298],[299],[300],[301]. However, Pajkos[74] reported that biofilm was
demonstrated with scanning electron microscopy in a single culture-negative sample.
Interestingly was the fact that extensive amorphous biological deposits were observed
99
with scanning electron microscopy, even in the absence of bacterial structures.
Moreover, because of the low pathogenicity of coagulase-negative Staphylococci and
the existence of microorganisms in a dormant phase within the biofilm around the
implant, capsular contracture does not usually clinically manifest until some remote
time after placement of mammary implants[74],[298],[299],[300],[301]. For all these reasons, in
a pre-clinical study, the authors did not consider the biofilm investigation. All the
methods to biofilm investigation are very expensive, are not routinely used and the
follow-up period should be really longer.
We sacrificed the rabbits at 2 and 4 weeks to study the capsule formation and try
to understand how it is possible to model wound healing formation[145]. Our study
demonstrated very similar wound healing results at 2 weeks among all the groups,
consistent with Adams and Marques et al.[84] report (Publication I). Our results differed
from Adams and Marques et al.[84] study where intracapsular pressures were increased
with fibrin glue application while in our results intracapsular pressure decreased. This
may be explained in part as our data were collected at 4 weeks while Adams and
Marques et al.
[84]
data examined more mature capsules at 8 weeks. On other hand, in
this previous study Adams and Marques et al.[84] applied an autologous fibrin glue of
unknown fibrin concentration into the implant pocket while in this experimental design
we sprayed a commercial fibrin product widely studied and used in clinical practice
(Tisseel/Tissucol®)
in
Europe
and
USA
to
reduce
polypropylene
meshes
adhesions[252],[302]. To the best of our knowledge, this is the first report examining
capsular formation with a commercial available fibrin product (Tissucol/Tisseel®). In
addition, this is the first study with investigation of bacterial contamination from
rabbit’s skin and operation room air.
100
The authors performed the study on a New Zealand white rabbit animal model,
an extension of Adams et al.[84] study, with the capacity to support four tissue
expanders, which is impossible in mice. There are limited reports with the use of
porcine.
One limitation of this study was the use of tissue expanders to measure the
pressure directly using ports, instead of commercial silicone breast implants with ports,
not available in this size. One strength of this study was the statistical analyses of these
data among the 4 groups using the CHAID method, modeling a single variable among
multiple variables.
Other parameters considered to be addressed in future studies: longer follow-up
time period; breast implants sprayed with fibrin (Tissucol/Tisseel®); focus on fibrosis
that may influence or modulate capsule contracture.
STUDY 3
Significant results were demonstrated in each of the experimental groups. In the
Fibrin group the data showed significantly decreased intracapsular pressures and
capsular thicknesses without any capsule rupture, compared to the Control and the
CoNS groups. For the Fibrin and the Control groups, decreased intracapsular pressures
were correlated with thinner capsules. A mixed type of inflammatory cells was the most
common finding for both Fibrin and Control groups. In the Fibrin group, loose or dense
≤ 25% connective tissue was observed compared to the Control and the CoNS groups
that had dense >25% connective tissue. In the Fibrin group, negative or mild
angiogenesis was observed compared with the Control and the CoNS groups with
moderate or high angiogenesis. No significant differences regarding fusiform cells
density were observed between the Fibrin and Control groups.
101
In the CoNS group, increased capsular thickness was measured compared to the
Control group. A polymorph type of inflammatory cells was the most common
observation in the CoNS group, significantly different from the Control group.
Regarding fusiform cells density, connective tissue, organization of the collagen fibers
and angiogenesis, similar results were observed for both CoNS and Control groups.
Similar bacterial isolates were observed among all the study groups, regarding
either implants or capsules. Implants were 2.7 times more frequently infected than
capsules. The predominant isolates were coagulase-negative Staphylococci, which were
present 3.8 times more in implants compared to capsules. There was no significant
association between microbiological and histological data. Bacteria isolates from
rabbit´s skin were similar to those isolated from capsules and implants. As expected, the
predominant isolate in rabbit´s skin, as in implants and capsules, were coagulasenegative Staphylococci. Unexpectedly, Micrococcus spp. were isolated from rabbit´s
skin specimens, operating room air samples and from one rabbit; in this specific rabbit,
Micrococcus spp. were detected on the capsule, but not on the implant surface, and this
capsule did not develop capsular contracture. Interestingly, on the contrallateral implant
in the same rabbit, a Micrococcus spp. isolate was detected on both implant surface and
capsule which was associated with clinical Baker grade IV capsule contracture
development (Figure 11). To the best of our knowledge, this is the first report that
shows a direct association between the presence of Micrococcus spp. and clinical
capsule contracture, in a rabbit model. Fungi were isolated from the operation room air
samples but not from the rabbit´s skin, capsules or implants. Even with similar bacteria
types observed among all the groups, regarding implants or capsules, fibrin still
modulates the capsule formation.
102
Our results support the probable role of fibrin as an agent that may modify
capsule formation and subsequent capsule contracture, with decreased capsule
thicknesses and pressures, loose or dense ≤ 25% connective tissue and negative or mild
angiogenesis. The decrease of intracapsular pressure correlating with thinner capsules
was also consistent with other clinical contracture reports[125],[126],[144],[303]. In addition to
these results, the dense connective tissue and increased angiogenesis related with
capsular contracture has already been demonstrated in other reports as achieved in our
Control and CoNS groups[126],[129],[296]. The organization of the collagen fibers (parallel
or haphazard) in capsular contracture is controversy; our results are similar to the study
of Karaçal et al.[144].
The cytokine transforming growth factor beta 1 (TGF-β1) is a central mediator
of fibrosis[304],[305],[306]. Some reports focused on fibrin properties for enhanced wound
healing by the reduction of collagen extracellular matrix and decreased TGFβ1[157],[162],[249],[250]. TGF-β1 inhibitor peptide was significantly effective in achieving a
reduction in fibrosis in silicone breast implants[297]. The use of fibrin-containing
preparations (Tisseel® and Vi-Guard®) allow the closure of dead-space and
approximation of the skin flaps, and it is argued that fibrin-containing tissue adhesives
produced such a dense architecture that angiogenesis and vascular ingrowth were
inhibited[251].To the best of our knowledge, this is the first pre-clinical study with a
commercial fibrin compound (Tissucol/Tisseel®), sprayed to a textured silicone breast
implant.
According to our results, bacterial infection of breast implants was more
common than capsules infection and the predominant isolates were coagulase-negative
Staphylococci. This is consistent with the fact that coagulase-negative Staphylococci, a
commensal bacteria of the skin, are the predominant cause of biomaterial-associated
103
infection, commonly mediated by the formation of biofilms[298],[299],[300],[301],[307],[308]. The
major pathogenicity is related to extensive biofilm formation on solid surfaces, which is
extremely difficult to treat with antibiotics, thereby necessitating invasive procedures to
remove the infected tissue or devices[309],[310],[311]. A strong correlation between the
presence of biofilm (particularly by S. epidermidis) and the presence of significant
capsular contracture were also reported[74]. They assumed that biofilm on the outer
surface of the implant, once established, acts as a focus of irritation and chronic
inflammation, leading to accelerated capsular contracture[74]. However, our results are
contradictory to this report[74]. In the study by Pajkos et al.[74], the rate of recovery
bacteria from the implant surface was lower than the rate of recovery from the capsule
surface, but the authors explain that there was a greater sensitivity in detecting bacterial
growth on capsules.
The clinical contracture Baker grade IV developed in one implant had the
thickest capsule (Figure 11) among all capsules studied and was unusual as the
contracture developed quickly with an acute inflammation. Histological evaluation of
fibrosis in this capsule contracture revealed dense 25-50% connective tissue, haphazard
collagen fibers and moderate angiogenesis. Unexpectedly, both the capsule and implant
were infected only with Micrococcus spp., a low pathogenic agent. As far as we know,
there are few reports concluding that Micrococcus spp. may have a true etiologic role in
infection[312] and mediated by formation of bacterial biofilms[313],[314].
Our fibrin results are contradictory to our previous report[84] (Publication I), but
consistent with another pre-clinical study[315] (Study 2; Publication III). This may be
explained as in this previous published study[84] (Publication I), where we applied an
autologous fibrin glue of unknown fibrin concentration into the implant pocket while in
this experimental design we sprayed a commercial fibrin product widely study and used
104
in clinical practice (Tisseel/Tissucol®) in Europe and USA to reduce polypropylene
meshes adhesions[252],[302], to reduce the incidence of posterior spinal epidural adhesion
formation[236], and to reduce the recurrence rate of pterygium after surgery[242]. Another
explanation was the application mechanism (manual with a syringe versus sprayed). A
previous study found that a thin layer of glue is preferable to a thick one[316]; a thin layer
of fibrin glue may support the healing process, whereas a thick layer of adhesive
inhibits skin graft healing[317]. Moreover was the fact that in this study capsule pressure
was measured directly in tissue expanders, to achieve more accurate results
[144]
. The
fibrin glue is used by its properties as a hemostatic agent[318]; for enhanced wound
healing by the reduction of collagen extracellular matrix and decreased TGF-β1
(mediator of fibrosis)[157],[162],[249],[250]; to prevent adhesions[252],[302];
ophthalmology[237],[238],[239],[240],[241],[319];
widely use in
used as a drug delivery system such as
antibiotic[320]; and our preclinical animal model results, make fibrin glue a promissing
agent to prevent capsular contracture. Furthermore, fibrin glue was already used in a
clinical model after breast augmentation as a drug delivery system[244].
The limitation of this study was the use of one tissue expander per rabbit just to
measure the pressure directly using port. To correlate intracapsular pressure from tissue
expanders with histological and microbiological results from breast implants, we
performed statistical analyses that revealed no significant differences in histological and
microbiological results between breast implants and tissue expanders. However, silicone
breast implants with ports 90 ml size would be better to achieve more accurate results
but are not commercially available. One strength of this study was the statistical
analyses of these data among the 3 groups using the CHAID method, a sophisticated
algorithm used in many other disciplines, adjusting the probability of a single variable
among multiple variables.
105
Possible future studies would include: 1) a prospective clinical study comparing
a women control group with a experimental group with Tissucol/Tisseel® sprayed to a
silicone
breast
implants/pocket,
with
a
follow-up
period
longer
than
42
months[321](Study1; Publication II); and 2) analyze S. epidermidis and Micrococcus spp.
biofilm development in a pre-clinical study with silicone breast implants with ports
sprayed with Tissucol/Tisseel® and infected with bacteria
STUDY 4
In this study, we report the development of capsular contracture in a rabbit
model associated with chitosan. All Chitosan group implants had clinical Baker grade
III/IV breast contractures with significantly thicker capsules than non-treated implants.
Chitosan exposed capsules were opaque, stiff and resistant to cutting and considerable
shrinkage, and folding of the implant surfaces were observed that may indicate the
constricting nature of fibrous implant capsules. Control group had thin capsule and
loose or dense ≤ 25% connective tissue compared to the dense > 25% connective tissue
observed in the Chitosan group. This is consistent with the fact that the major
component of chitosan, glucosamine, forms cartilage tissue and is also present in
tendons and ligaments[146]. Collagenous layer of granulation tissue is increased by
chitosan applications; according to this finding, chitosan may stimulate fibroblast
proliferation and extracellular matrix production[287]. Chitosan induced an accelerated
wound
healing
process
which
increased
TGF-β1
responsible
for
several
proinflammatory regulatory influences, including cell migration, granulation tissue
formation and increased collagen production[277] and, recognized as a central mediator
of fibrosis[155].
106
A mixed or polymorph type of inflammatory cells was the most common finding
in all rabbit capsules and inflammation intensity was moderate or mild in all capsules
which was expected as chitosan is chemoattractant for neutrophils[220],[322]. Chitosan
enhances the function of inflammatory cells such us polymorphonuclear leukocytes
(PMN), macrophages, fibroblasts (production of IL-8), angioendothelial cells[287] and
LMWC has a systemic effect[283]. Apoptotic cells and necrosis were observed strongly
in Chitosan capsules which were consistent with other reports[323],[324].
Statistical analyses revealed no significant differences in the frequency of
culture positivity and types of bacteria among all the groups. Interestingly, no
significant associations between microbiological and histological data were observed in
any group. Similar bacterial isolates were cultured from rabbits’ skin and air samples
and the predominant isolates were coagulase-negative Staphylococci. The antimicrobial
activity of chitosan and its derivatives against several bacterial species has been
recognized and considered as one of the most important properties linked directly to
their possible biological applications[217],[218],[219],[220]; however, the new interest on
chitosan as a drug deliverer such us antibiotics questioned the high efficacy of chitosan
alone as an antibacterial agent[325],[326],[327],[328].
This study supports that capsular
contracture formation was not the result of bacterial infection alone, in contrast to the
infectious hypothesis which has been championed and consistently supported by
Burkhardt[61],[63],[84].
To gain insight into the inflammatory process, the major biomarkers, TNF-α and
of IL-8, were measured. This is the first report examining extracellular levels of IL-8
and TNF-α in a breast capsule implant environment. Microdialysate levels of IL-8 were
decreased (p< 0.05) in the Chitosan group compared to the Control group. No
significant differences in the microdialysate levels of TNF-α were observed among the
107
groups. In the Control group, a correlation between IL-8 and TNF-α was observed; no
significant correlation between IL-8 and TNFα levels were observed in the experimental
groups.
We originally hypothesized that serum concentrations of the inflammatory
mediators would be significantly increased in the Chitosan group due to the expected
greater inflammatory response with Chitosan as this molecule promotes the production
of IL-8[287]. These data did not support the hypothesis but were consistent with Tilg et
al.[329] study which reported increased IL-8 and TNF-α levels in bacterial infection and
decreased
IL-8 and TNF-α levels in acute rejection. Interestingly, we now report
clinical Baker grade III/IV breast capsule contractures in all rabbits exposed to chitosan
associated with polymorph and mixed inflammation and not due to a bacterial infection.
Not all Chitosan implants were infected and IL-8 and TNF-α were decreased in the
Chitosan group. Molecular regulation of IL-8 production has been studied in vitro and
TNF-α has proven to be a major regulatory molecule. It is not surprising that in vivo IL8 and TNF-α serum levels were also significantly correlated in the Control group.
Correlation between IL-8 and TNF-α was well established in the case of bacterial
infection, less pronounced in cytomegalovirus hepatitis and not apparent in acute
cellular liver rejection episodes. Lack of correlation in acute rejection was also
associated with low levels of IL-8[329]. This suggests that in contrast to bacterial
infection, countering cytokines may be active in capsular contracture (at least promoted
by chitosan), and down-regulating IL-8 transcription and/or translation. So far, no
reports exist on production and regulation of IL-8 in capsular contracture. Recent
studies have additionally demonstrated that COS displayed anti-inflammatory properties
in immunocytes including the inhibition of nitricoxide, the down-regulation of IL-6 and
TNF-α and the increase of cell viability of neutrophils[330],[331]. Additionally, IL-8 was
108
induced by a wide range of stimuli, including lipopolysaccharide (LPS), a component of
the outer membrane of Gram-negative bacteria and TNF-α. The study by Lund et
al.[190] concluded that LPS induced IL-8 release in monocytes, while TNF-α was a good
inductor of IL-8 in PMN. In the chitosan contracture model, we had decreased levels of
IL-8 and it was possible to conclude that there was no Gram-negative bacteria infection
to induce IL-8. On the other hand, chitosan increased the production of TGF-β1[277], a
central mediator of fibrosis; the degree of capsular contracture is directly related to an
increased level of TGF-β[125]. Even with contradictory studies about the role of TNF-α,
Moritomo et al.[332] concluded that TNF-α played a pivot role in the maintenance of
hemostasis and tissue repair by inhibiting TGF-β1.
Our data support the theory that chitosan initiates capsular contracture response
due to a toxic local effect that resulted in an impaired wound healing response. An
earlier series of pilot studies were performed with much higher levels of chitosan (data
not shown). Using similar experimental protocol in the rabbit model, implants exposed
to 25.0 mg/mL levels were used. The majority of animals expired within a short time
period; surviving animals had decreased weight (15-25.8%) compared to baseline body
weights with leucocitosis and decreased hemoglobin. At autopsy, fat biopsies were
atrophied and liver specimens had lymphoid infiltration in portal spaces. We report
toxicity with 25.0 mg/mL of implanted LMWC per rabbit. The study design was
modified to test decreased chitosan levels that were not systemically toxic to the
animals. In the reported data, all animals were clinically healthy.
Literature data
reporting general toxicity testing for chitosan is limited[276] and our results are consistent
with the few papers about chitosan toxicity[283],[284],[285],[286],[287].
In several important studies[73],[77] the same rabbit had different implants.
Darouich et al.[73] with the objective to examine in vivo the antimicrobial efficacy of
109
minocycline/rifanpin-impreganated saline-filled silicone implants, used the same rabbit
to place 4 implants (2 antimicrobe-impregnated and 2 control implants were placed in
each rabbit). In the study by Shah et al.[77], with the objective to examine in vivo the
infectious
hypothesis,
each
rabbit
underwent
a
Staphylococcus
epidermidis
contaminated implant and a control implant. However, due to the systemic influence of
chitosan, the use of 3 different implants in the same rabbit, in our study, obviously may
confounded the results. To clarify this issue, a Control limb study was performed (data
not shown) and compared with the Control group of this study. Using similar
experimental protocol, 10 rabbits were implanted with 2 textured breast implants.
Interestingly, on the Control group from this study the capsular thickness was lower
than in the Control limb group (0.81 ± 0.21 mm) (p = 0.001). No significant differences
were observed regarding the intensity of inflammation, characteristics of connective
tissue (either loose or dense), fusiform cells density and angiogenesis between the
groups; significant differences were observed with respect to the type of inflammatory
cells, with mixed type of inflammatory cells in 54.5% of the Control group of this study
and mononuclear type of inflammatory cells in 55.6% of the Control limb group (p =
0.017); significant differences were observed in the organization of the collagen fibers,
which were arrayed in sequence in the Control group of this study and haphazard in the
Control limb group (p = 0.007). Statistical analysis revealed no significant differences
in the type of bacteria and frequencies between the control group of this study and the
control limb group. A decreased levels of IL-8 (p = 0.016) and TNF-α (p = 0.001), were
observed in the Control group of this study when compared with the Control limb
group, which prove the systemic influence of chitosan.
In the discussion of our previous paper[84] (Publication I), Burkhard considered
that if a rabbit model must be used for research, a more appropriate model was that
110
reported by Shah et al.[77],[288] who used bacterial contamination to produce contracture.
In that study[77], 16 New Zealand white rabbits underwent each one, a Staphylococcus
epidermidis contaminated implant and a control implant. The capsules were dissected at
2, 4, 6 and 8 weeks. Capsules on the contaminated side were 2 to 3 times thicker than
those on the control side, and did not change thickness with time. Capsules on the
contaminated side consisted of densely packed longitudinally oriented thick bundles of
collagen fibers; there was a large cellular infiltration with leukocytes and macrophages.
In contrast, the capsules on the control side were thinner and consisted of loosely
organized connective-tissue fibers predominantly parallel to the prosthesis surface.
Bacteriologic cultures on the contaminated side consistently isolated Staphylococcus
epidermidis with occasional diphtheroides, while the control side showed no bacterial
growth. As Prantl et al.[333] we believe that subclinical infection with chronic
inflammation represents one of the possible important reasons for the development of
capsular contracture. We also hypothesize that all possible causes of fibrosis result in
the common key factor of pathological response with the development of chronic
inflammation. Prantl et al.[333] included only those implants with high gel cohesiveness
(third-generation implants); in these implants, the silicone filler presumably does not
leak from the shell into the tissue in case of implant rupture; surprising, in 67% of their
specimens, they detected vacuolated macrophages with microcystic structures
containing silicone, and in 54% of the specimens, the capsular tissue contained empty
spaces of varying sizes of silicone particles. It remains unclear whether these silicone
structures represented friction particules from the surface of the implant or particules in
the implant filler. Heppleston and Styles[334] study performed in vitro experiments
demonstrating that silica damages macrophages, which subsequently produces TGF-β1
which stimulates fibroblast to produce collagen. However, since the study by Shah et
111
al.[77], and as far as we know, even with many publications with infected implants, there
was no translation of the Baker classification in a pre-clinical model.
An infection-induced contracture limb study was performed (data not shown)
and compared with the Chitosan group of this study. Using similar experimental
protocol, 10 rabbits were implanted with 2 textured breast implants, each one with a
suspension of 100 microlitres of coagulase-negative Staphylococci (108 CFU/ml - 0.5
density in McFarland scale). Histologically, the average capsular thickness was 1.065 ±
0.287 mm in the infection-induced contracture limb group (CoNS group) and 2.746 ±
0.817 mm in the Chitosan group. Capsular thicknesses were found to be statistically
different among the two groups (p = 0.00003). A significant difference was also
observed regarding the type of inflammatory cells among the two groups (p = 0.021),
with a polymorph type predominant in the CoNS group, and mixed type predominant in
the Chitosan group. No significant differences were found between the two groups
regarding the intensity of capsule inflammation. Significant differences in the
angiogenesis were found between the CoNS and Chitosan groups (p = 0.004), with
equally absent/mild and moderate/high in the CoNS group but only high in the Chitosan
group, as well as in the synovial metaplasia (p = 0.043) which was always absent in the
Chitosan group but present in some cases of the CoNS group. However, no significant
differences were found between the two groups regarding the characteristics of the
connective tissue, organization of the collagen fibers (parallel or haphazard) and
fusiform cells density. Histologically, this type of capsular contracture induced by
chitosan is different than those induced by infection, in some aspects: 1) the capsule was
thicker; 2) the mixed type of inflammatory cells was predominant; 3) the angiogenesis
was high; 4) the synovial metaplasia was absent. This study reported to science a preclinical non-infectious model of capsular contracture and further studies are necessary.
112
We sacrificed rabbits at 4 weeks to study early capsule formation and to
understand how it is possible to model wound healing formation[145]. The point is well
taken for longer time periods and is currently planned for future experiments. However,
long term differences in capsule structures under these experimental challenges do
result from different wound healing trajectories from day 0. Our strategy was to
examine these early differences with methods that were sensitive to detect histological
or biomarker changes. There is no answer how long enough is necessary in a preclinical model. In a clinical model the authors propose a follow-up period longer than
42 months[321] (Study 1; Publication II). However, it might be expected that the finding
of a dense collagenous capsule would increase with time, reflecting a continued
stimulus toward a fibroplasia and ultimately collagen remodelling[335],[336],[337].
The weakness of this study was the relatively small size and the lack of capsule
immunohistochemestry detection of IL-8 and TNF-α in tissue specimens. Nevertheless,
the release of IL-8 and TNF-α into the circulation represented a “spillage” of factors
rather than a direct signal driving inflammation and leukocyte recruitment; the use of
microdialysis was appropriate for determining tissue concentration of cytokines such us
IL-8 and TNF-α. Because of the proximity of the sampling site to the source of the
cytokine, microdialysis may provide a means of sensitively detecting relative changes
of inflammatory mediators’ concentration with experimental treatments.
Possible future studies would include: 1) silicone breast implants with ports (to
measure the capsule pressure directly) impregnated with low molecular weight chitosan
(LMWC) implanted per rabbit; detection of IL-8, TNF-α, TGF-β1 and determination of
fibrosis index; 2) With the same protocol analyze silicone breast implants with ports
impregnated with low molecular weight chitosan (LMWC) and sprayed with
Tissucol/Tisseel®[315],[338] .
In summary, a capsular contracture animal model was
113
observed when implants were impregnated with chitosan and not due to a bacterial
infection. This report suggests a new approach of studying capsular contracture using a
pre-clinical animal model.
STUDY 5
The capsule is composed by a layer of fibrous dense connective tissue[339], and is
an integrant part of the wound healing process. To understand the formation of this late
complication, and the potential therapeutic roles of both pharmacological and non
pharmacological approaches, it is crucial to know the physiological mechanisms that are
behind this process.
Wound healing has been divided into three distinct phases: inflammation,
proliferation and maturation[340]. The first phase of wound healing, which courses
immediately upon injury through day 4 to 6, is characterized firstly by hemostasis, an
important event that serves as the initiating step for the healing process; and an
inflammatory response. The second phase of wound healing (proliferative phase) is
characterized by epithelization, angiogenisis and provisional matrix formation, and
courses from day 4 through 14, overlapping the phase 1 and 3. Fibroblasts and
endothelial cells are the predominant cells proliferating during this phase. Maturation
and remodeling (phase 3), occurring from day 8 through 1 year, is characterized by the
deposition of collagen in an organized and well-mannered network[145].
As seen before, corticosteroids are known to have an important role on
modelling wound healing, as they can stop the growth of granulation completely, the
proliferation of fibroblasts, diminish the new outgrowths of endothelial buds from blood
vessels and stop the maturation of the fibroblasts already present in connective
tissue[227].
Also when administered early after injury, corticosteroid delay the
114
appearance of inflammatory cells, fibroblasts, the deposition of ground substance,
collagen, regenerating capillaries, contraction and epithelial migration[228]. As so,
steroids can have an important role in CC formation, in both early and late phases of
fibrous phase formation.
The efficacy of triamcinolone on treating and preventing CC in women has been
reported[265], [266]. However, this still represents an off-label practice and further studies
are required to validate the efficacy of this approach. Both works have limitations: are
non-randomised, with no control group, with a limited follow-up period[265],[266] and
none of them have, as an objective, to comprove which is the mechanism of action of
this compound in capsular contracture formation.
A comprehensive understanding on the effects of this compound on the
mechanisms of capsular formation; a knowledge on the systemic side effects and
potential adverse events are, in the authors opinion, crucial for the improvement of TA
in clinical activity.
This study arises as the first one analyzing the impact of TA in early capsule
formation. The authors examined the effects of TA on pressure, histological,
microbiological and immunological characteristics of capsules, in an animal model, in
order to understand the role of this steroid in early capsule formation, and the possible
role in the prevention of CC.
In our study, TA was found to decrease capsular thickness both on macroscopic
and microscopic examination, when compared to the Control group. These findings
were also associated with decreased inflammation and angiogenesis, as it was expected
as steroids are anti-inflammatory drugs, capable of delaying the appearance of
inflammatory cells and diminish the proliferation of endothelium from blood vessels[227]
and regeneration of capillaries[228]. Although no significance was found in the
115
intracapsular pressure between groups, it was observed a tendency to lower pressures
(and no capsule rupture during the pressure measurement) in the Triamcinolone group
when compared to the Control group (Figure 14). Also both cytokine markers (IL-8 and
TNF-α) were lower in the Triamcinolone group, even without statistic significance. No
significant differences were observed in fusiform cells densities, connective tissue and
organization of the collagen fibers. Taken together, these results suggest that washing
the pocket intraoperatively with TA has a role on capsule formation, and might prevent
CC.
As Caffee et al.[264], we were not able to observe a significant decreased capsular
pressure in the group treated with triamcinolone in the time of implant placement.
However in our study we go further and we analyzed not only the pressure, an
unquestionable indicator of capsular contracture, but also other characteristics that are
related with the formation of this pathology, as a continuous process. The breast capsule
begins being formed after implant placement, however, in the clinical practice, the
contracture is a late complication, and a follow-up as longer as 42 months (Study 1;
Publication II)[321], is required to the diagnostic of this entity. In preclinical models,
there is no consensus on the timing for sacrifice and timing for representative stages for
capsule formation.We were not able to observe significant differences on pressure
between the groups, probably because we sacrificed animals too early to the complete
development of this complication. However, we were able to observe the early
alterations that are not characteristic of CC as thinner and more transparent capsules on
macroscopic and microscopic evaluation, and decreased inflammation and angiogenesis.
It might be expected and is reasonable to assume that a more dense collagenous capsule
with increased thickness would be present with longer incubation times, reflecting a
116
continued
stimulus
toward
a
fibroplasia
and
ultimately
collagen
remodelling[336],[335],[337].
Caffee et al. reported in both preclinical[264] and clinical[265] studies that
triamcinolone injected postoperatively was able to eliminate CC and prevent the
recurrence of this condition. Those findings are confirmed by Sconfienza et al.[266], that
was able to demonstrate that US-guided injection of triamcinolone acetonide in the periimplant pouch of women with augmented or reconstructed breast affected by Baker
grade IV CC is effective in reducing capsular contraction. Both authors concluded that
triamcinolone was effective in the late stages of capsule formation. With our study we
were able to observe that triamcinolone is probably not only effective when injected
postoperative, but it also as a role in the early phases of the development of capsular
contracture.
In a previous report (Study 3; Publication 4)[338] using the same protocol, the
authors were also able to find another compound, fibrin (Tissucol/Tisseel), that was
associated with a lower incidence of CC when sprayed in the pocket/implant during
surgery and more effective in the early phases of wound healing than in later phases. It
was found that fibrin[338] , was able to decrease intracapsular pressures when compared
to Control (p  0.001 - data not shown), and the capsular thickness were decreased (0.47
± 0.129 mm) (p  0.001) as in Triamcinolone group.
TNF-α plays an important role in the wound healing process: it is produced by
activated macrophages, platelets, keratinocytes, and other tissues and it stimulates
mesenchymal,
epithelial,
and
endothelial
cell
growth
and
endothelial
cell
chemotaxis[341],[342]. During the inflammatory phase, it is one of the main responsible for
neutrophils drawn into the injured area[343], macrophages generation of NO[344] and
damaged extracellular matrix digestion by matrix metalloproteinase[345]. During the
117
second phase TNF-α upregulates KGF gene expression in fibroblasts, upregulate
integrins, a matrix component that serves to anchor cells to the provisional matrix,
stimulates epithelial proliferation[341] and is also a potent promoter of angiogenesis.
TNF-α is known to be a growth factor for normal human fibroblast, and promotes the
synthesis of collagen and prostaglandin E2. IL-8, enhances neutrophil adherence,
chemotaxis, and granule release; and enhances epithelization during wound
healing[341],[346]. TNF-α levels were reported to be markedly elevated in fibrotic diseases
as liver fibrosis, and is considered a mediator of fibrosis such as TGF-β1[346]. Moritomo
et al.[332] concluded that TNF-α played a pivot role in the maintenance of hemostasis
and tissue repair by inhibiting TGF-β1. We were not able to find significant differences
in IL-8 and TNF-α levels, although decreased levels were observed in the group treated
with triamcinolone, possibly reflecting a role of this drug in modulation of wound
healing process and fibrotic response in the presence of the implant. More studies, with
longer follow-up and increasing doses of the compound are needed to confirm this data.
On the other hand, a significant correlation was also found between IL-8 and
TNF-α in both groups. This was not unexpected as correlations between IL-8 and TNFα with bacterial infections have been reported[329]. We did not find any differences in the
microbiology cultures between groups but further studies are necessary to clarify if
triamcinolone increases the risk of infection.
With fibrin it was observed a significant decreased on TNF-α (140.9 ± 165.9
mg/ml) and IL-8 (23.9 ± 43.4 mg/ml) levels (p = 0.003; p = 0.048) supporting the
possible role of this compound in the early capsule formation and in the reduction of
collagen extracellular matrix. No correlation between IL-8 and TNF- α was observed in
the Fibrin group which suggests a possible antibacterial role of fibrin[338].
118
The main limitations of this study are: 1) inappropriate dosage in this model
system; rabbits have much faster basal metabolic rates than human, and as such, it is
presumed that rabbits have shorter drug half-lives[268]; 2) unknown pharmacokinetics of
triamcinolone in the capsule pocket and subsequent metabolism, although triamcinolone
modelling may be based on systemic steroid modelling[347]; 3) short follow-up, as
capsular contracture usually takes more than four weeks on developing; and 4) the use
of one tissue expander per rabbit to directly measure internal expander pressures using
the port. Silicone breast implants with ports with a 90 ml volume capacity would be
optimal to achieve more accurate results but are not commercially available. In addition,
the preclinical model would not support the use of multiple large expanders or implants
over long time periods.
Our data support future studies examining triamcinolone as a potential agent on
preventing CC.
Possible future studies may include: 1) pre-clinical study with silicone breast
implants with ports (to measure the capsule pressure directly) with introduction of 1.5
ml (60 mg) of triamcinolone-acetonide into each implant pocket and sacrifice the
animals at a much longer time point with detection of IL-8, TNF-α, TGF-β1 and
determination of fibrosis index; and 2) with the same protocol, assess the effects of of
saline or other vehicles of triamcinolone-acetonide on pressure/volume curves. We
believe that a pre-clinical study higher dose of triamcinolone-acetonide introduced into
the implant pocket and a longer follow-up time period will support the growing body of
evidence that triamcinolone-acetonide mitigates capsular contracture.
In summary, our results suggest that triamcinolone has a role in early capsule
formation, and it may have a role in the prophylactic management of this complication.
Obviously, it role is centred on the management of the factors related to wound
119
healing[124], and it is important to exclude a deleterious role in the factors related to
infection that are also known to increase CC[62],[64],[79],[21],[206]. The clinical use of
triamcinolone-acetonide may prove to be a reliable and safe intraoperative method to
prevent capsular contracture in women undergoing breast implants. The ultimate goal is
to translate these preclinical results to the clinic as these finding may help not only
patients with breast implants, but to all patients with any device in which capsule
contracture around that device may lead to an adverse clinical event.
120
6. Conclusions

The authors for the first time report no association between capsular contracture
and menopause or estrogen status. (Study 1; Publication II)

Our data suggest that grade II subjects should be included in a capsule contracture
analyses and a follow-up period longer than 42 months (3 years and 6 months) should
be considered. (Study 1; Publication II)

The authors document a pre-clinical capsular contracture with thicker capsule,
mixed or polymorph inflammatory cells, dense >25% connective tissue, haphazard
collagen fibers or arrayed in sequence, moderate or high angiogenesis. (Studies 3 and 4;
Publications IV and V )

It is not appropriate to sacrifice the animals at 2 weeks. (Study 2; Publication III)

Bacteria from rabbit´s skin and operation air were similar to those from removed
capsules, tissue expanders and breast implants, but, the fungi were just isolated in the air
samples. (Studies 2, 3, 4 and 5; Publications III, IV, V and VI)

Interestingly, in the Fibrin (Tissucol/Tisseel®) group mixed type of inflammatory
cells were correlated with decreased intracapsular pressures, however if infected by
Staphylococcus the intracapsular pressures increased. Our results suggest the role of
fibrin (Tissucol/Tisseel®) in preventing capsular contracture; and that the bacterial
colonization of mammary implants may be partially responsible for capsule contracture,
and coagulase-negative Staphylococci may play a large role. (Studies 2 and 3;
Publications III and IV)

The role of Micrococcus spp. in the pathogenesis of capsular contracture deserves
further study. (Study 3; Publication IV)

Based on the Blood and the Thrombin (FloSeal®) groups results we may conclude
121
that an active hemostasis is indispensable to prevent capsular contracture, although
unnecessary with a hemostatic commercial product. (Study 2; Publication III)

The fibrin (Tissucol/Tisseel®) benefits enhance wound healing reducing capsular
contracture and the minor adverse events observed make this drug an attractive
alternative for use in women undergoing breast implantation. (Studies 2 and 3;
Publications III and IV)

A capsular contracture animal model related with chitosan and not due to an
infection. (Study 4; Publication V)

The use of microdialysis is appropriate for determining the concentration of
cytokines such us IL-8 and TNF-α and could provide a means of sensitively detecting
relative changes of inflammatory mediators’ concentration with experimental
treatments. (Study 4 and 5; Publication V and VI)

Our data support the theory that chitosan initiates capsular contracture response
due to a toxic local effect that resulted in an impaired wound healing response. (Study
4; Publication V)

Triamcinolone-acetonide during breast implantation influences the early capsule
formation and may reduce capsular contracture. (Study 5; Publication VI)

Capsular contracture is multifactorial including any bias which promotes a
subclinical toxic effect. (Studies 2, 3 and 4; Publications III, IV and V)
122
Financial disclosure and products page
The present study was carried out at the Faculty of Medicine, University of
Oporto, Portugal (Department of Experimental Surgery and Department of
Microbiology), the Centro Hospitalar of São João, Oporto, Portugal (Department of
Plastic and Reconstructive Surgery and Department of Pathology), the Faculty of
Sciences, University of Oporto, Portugal (Department of Chemistry), the Biotechnology
School, University of Oporto, Oporto, Portugal and the UT Southwestern Medical
School at Dallas, Texas, USA (Department of Plastic Surgery and Research, Nancy L.
& Perry Bass Advanced Wound Healing Laboratory).
This thesis was partially supported by grants from the Fundação Ilídeo Pinho
and Comissão de Fomento de Investigação em Cuidados de Saúde Daniel Serrão.
Implant devices were supplied by Allergan Medical Company, Ireland and
Expomedica, Portugal.
Tissucol/Tisseel® and FloSeal® were supplied by Baxter Healthcare
Corporation, Vienna, Austria.
There is no conflict of interest.
123
124
Acknowledgements
Starting my academic life and research under the supervision of two outstanding
scientists, has truly been a privilege. First and leading, I owe my sincere gratitude to
Professor José Amarante. In 1998, after the National Medical Exam (which allows us to
choose a Residency Program based on ranking), I prepared a list of several surgical
specialities and different departments. One by one, from each hypothesis, I spoke with
the respective Director, a Specialist and a Resident. I had to be sure of that choice as it
would change the rest of my life. The interview with Professor José Amarante was
decisive. I remember that day so well, because I was so nervous: he was (and still is so
far) the Big Boss in Plastic Surgery! It wasn’t the interview that I had anticipated but
instead an engaging and interesting conversation. During 1 hour, Professor José
Amarante outlined all of the Plastic Surgery themes, the opportunities and challenges as
well as the duties, hard work, academic expectations, his demands from a resident, and
as a result, he removed all my fears. When I questioned him about the possibility of a
foreign fellowship (it was the key of my decision), he answered: “You have to; at least
six months!”. He made my day; he “decided” my professional life! Since that day,
Professor José Amarante has held those same high standards! For me, he is a pushing
mentor... pushing me up and more and more, all the time! I express my gratitude with
all my heart! Sometimes, it seems that our lives are in the form of a circle ... this day
was reiterated. In 2002, I decided to travel for three months to do a microsurgery
fellowship in Taiwan with Professor Fu-Chen Wei. Regarding the remaining 6 months,
it would be hard to achieve because I wanted to experience both clinical research with
basic science and facial aesthetic surgery. The Department of Plastic Surgery at
University of Texas Southwestern Medical School at Dallas, Texas, USA has some of
125
the best Aesthetic Facial Surgeons and is famous for resident’s mentorship, scientific
publications and has their own research lab - the Nancy L. & Perry Bass Advanced
Wound Healing Laboratory. I forwarded my Curriculum Vitae and a letter to Research
Professor Spencer Brown, and also a letter to Dr. Rod Rohrich. One week later, I was in
the operating room, in the middle of 2 surgeries, and I received a phone call from
Professor Spencer Brown, the Director of the Research Department of Plastic Surgery at
UT Southwestern Medical School in Dallas! He was so friendly with such
encouragement during this phone call (without knowing me at all!) which was, to me,
unbelievable! Once again, I was so afraid to disappoint, but accepted and started my
research projects with Professor Spencer Brown in 2003. His far-reaching vision, not
only in science, but also in life, has been a true inspiration to me.
After returning from Dallas in 2004, Professor Amarante introduced me in the
Faculty of Medicine, University of Oporto. I was and I´m so proud! Last year, 2010, in
the Times Higher Education World University Ranking, the University of Oporto was
classified in the 250th position of the best university in the world and the 106th in
Europe. I completed my Plastic Surgery Residency in February 2005. The next day,
Professor Amarante asked me to start a PhD thesis. I confess: I was exhausted! He
introduced me to Professor Acácio Gonçalves Rodrigues, the Director of the
Department of Microbiology. The microbiology study was crucial in this thesis. In the
first meeting with Professor Acácio Gonçalves Rodrigues, among a multitude of
students in the 3rd Exhibition of Science, Education and Innovation of University of
Oporto, he outlined this project and encouraged me strongly to go ahead! During this
entire project, the Department of Microbiology and Professor Acácio Gonçalves
Rodrigues provided faithful support. During our scientific discussions, he would
suggest new fantastic ideas. I presented them to Professor Spencer Brown who has
126
taken ownership of these studies as a true mentor. He is indeed an extraordinary teacher,
and his passion towards life and science are contagious. He is the most altruist person I
have ever met, but he firmly expects that others do their best as he does himself.
Without his guidance and continuous support, it would have never been possible to
make a bridge between Oporto and Dallas, which was the most grateful thing that has
happened to me! For our weekly meetings by phone, almost all my holidays working
with him in Dallas (since 5 in the morning!), being received in his home as a family
member, for meeting his wonderful wife Roxane and his daughter Keersten, for his
brightness in science, for his sense of humor, for the economic support… my deepest
gratitude and friendship! What such good memories I have of Dallas! It is now my
second home, difficult to survive without being there twice a year! It was an honor and
a privilege to learn not only about science from a great scientist, a master in
grantsmanship for economically supporting his research and team, but also about life,
family, sense of community and humanity! I am so blessed to be a part of such an
amazing family!
I present my gratitude to: Master Pedro Rodrigues Pereira for his tremendous
dedication and efficient work in the histological analysis of the samples; Professor João
Fernandes from the Biotechnology School, due to the impregnation of the breast
implants with chitooligosaccharide mixture and low molecular weight chitosan and his
expertise on this issue, and for whom I am grateful for teaching me so much; and Dr.
Luis Cobrado for his collaboration in the microbiology section.
I also present my gratitude to Professor Natália Cordeiro for her tremendous
effort in the statistical analysis. Professor Natália has been my friend for 20 years, and is
a professional at the highest level and dedication! However during this project, she was
127
operated on twice by neurosurgery. I will never forget that in the days following each
surgery, she wanted to discuss the statistical results and was concerned not to delay my
thesis! For the thousands of hours in her house (sometimes working all night), for her
incredible perseverance, brilliant mind, knowledge dependency and friendship, I express
my profound gratitude! Furthermore, I thank her for the friendship and care she offered
to me, particularly when I most needed, despite of her numerous tasks! This project
wouldn´t be the same without her. I also forward my thanks to Professor Aliuska
Helguera-Morales who also performed the statistical analysis and assisted me a great
deal and always reminded me of her mother land- Cuba! She has a precise and rational
mind, a high capacity of organization and her simplicity and lovely heart was always
available to help me and was an example of generosity!
I have a special gratitude to the veterinary surgeon, Fernando Carvalho, for his
incredible dedication to this project. The New Zealand white rabbits are very sensitive.
In study 2, he brought from his private clinic the anesthesia equipment. During the
entire research program, he was available and changed his schedules all of the time in
order to support this project. I would like to say thank you to the other members of the
Department of Experimental Surgery for assisting me in the care of my rabbits: Maria
José Neto; Pedro Leitão; Luis Bastos; and Maria José. The same gratitude goes to Nuno
Rego, who raised these special rabbits… for an instance I was afraid that I would have
to import them! To the Department of Microbiology, I would like to thank Anabela
Silvestre, Isabel Santos, Cristina Moura and Elisabete Ricardo. Related with the
implants devices and commercial products, I would like to thank them for their
tremendous help: Tom Powell, Luis Sogalho, Pedro Lopes and Luis Lopes.
128
At UT Southwestern Medical School at Dallas, my extreme gratitude to them for
their support: Dr. Rod Rohrich and Dr. Jeffrey Kenkel for the strong encouragement
during all these years. To Dr. William Adams, for the knowledge about this issue and
giving me the privilege of being a part of his team. To Dr. Jeffrey Kenkel, Dr. Michel
Saint-Cyr and Dr. James Richardson for their scientific experience, guidance and
revision of the papers. To Jiying Huang, Debby Noble, Donna Henderson for their
excellent assistance.
To the medical students who helped me in organizing much of this work: Lara
Queirós (current specialist in Ophthalmology), Rui Freitas (current resident in Urology),
André Santos Luís (current specialist in Stomatology), Mário Mendanha (current
resident in Plastic Surgery), and Nuno Lima (current specialist in General and Family
Medicine).
To the Plastic Surgery team I work with, for the encouragement and professional
help, with a special gratitude to my friend and current Director, Dr. Álvaro Silva and
my resident, Dr.ª Inês Correia Sá.
To my special friends and my godson Miguel for their emotional support, strong
encouragement and for understanding my absence time. I have the best friends in the
world!!!
I´m extremely grateful to my family: my mother for her kindness and freedom
love and to my father for his rationality and strong behavior, during my entire life; my
two brothers for the strong friendship and care between us; to my sisters-in-law, my
niece and goddaughter Inês, and my nephew Diogo that brought more happiness to my
family.
129
Finally, I would like to thank Maria de Lurdes for taking care of my son as he
belongs to her! She took care of my home and gave to Gustavo such love and peace,
and worked with him the rules he needs to grow up with respect and self confidence!
Moreover, she was available at any time of the day or night to come to my home to help
me! There is not enough money to pay for that! This serenity allowed me to work hard,
especially when it was outside of my home!
Last, but most important: my lovely son Gustavo. I am sorry when I was
working at night, or during the week-ends, or I was in Dallas, or I was so exhausted! I
know that he is only 3 years old and that it is impossible to explain to him so many
things, but so far I have concealed the worries and he has translated that in a funny
smile to my eyes! What a miracle power he has over me! There are no words in any
language to explain this huge feeling full of love and responsibility… in his little hand
he handles my heart! I have grown up listening to my parents saying that the best and
most difficult thesis in one’s life is the education of their sons, and, at least, they had to
be a step above the parents! They did an excellent job! This thesis is dedicated to
Gustavo! I had to work hard in all senses to give to him the best opportunities and be an
example! I will do my best… just his future will give me the big answer.
130
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150
Original publications
151
Publication
EXPERIMENTAL
A Rabbit Model for Capsular Contracture:
Development and Clinical Implications
William P. Adams, Jr., M.D.
M. Scott Haydon, M.D.
Joseph Raniere, Jr., M.D.
Suzanne Trott, M.D.
Marisa Marques, M.D.
Michael Feliciano, M.D.
Jack B. Robinson, Jr., Ph.D.
Liping Tang, Ph.D.
Spencer A. Brown, Ph.D.
Dallas, Texas
Background: Capsular contracture remains one of the most common complications involving aesthetic and reconstructive breast surgery; however, its cause,
prevention, and treatment remain to be fully elucidated. Presently, there is no
accurate and reproducible pathologic in vitro or in vivo model examining
capsular contracture. The purpose of this study was to establish an effective
pathologic capsular contracture animal model that mimics the formation of
capsular contracture response in humans.
Methods: New Zealand White rabbits (n ⫽ 32) were subdivided into experimental (n ⫽ 16) and control groups (n ⫽ 16). Each subgroup underwent
placement of smooth saline mini implants (30 cc) beneath the panniculus
carnosus in the dorsal region of the back. In addition, the experimental group
underwent instillation of fibrin glue into the implant pocket as a capsular
contracture–inducing agent. Rabbits were euthanized from 2 to 8 weeks after
the procedure. Before the animals were euthanized, each implant was serially
inflated with saline and a pressure-volume curve was developed using a Stryker
device to assess the degree of contracture. Representative capsule samples were
collected and histologically examined. Normal and contracted human capsular
tissue samples were also collected from patients undergoing breast implant
revision and replacement procedures. Tissue samples were assessed histologically.
Results: Pressure-volume curves demonstrated a statistically significantly increased intracapsular pressure in the experimental group compared with the
control group. The experimental subgroup had thicker, less transparent capsules than the control group. Histologic evaluation of the rabbit capsule was
similar to that of the human capsule for the control and experimental subgroups.
Conclusions: The authors conclude that pathologic capsular contracture can be
reliably induced in the rabbit. This animal model provides the framework for
future investigations testing the effects of various systemic or local agents on
reduction of capsular contracture. (Plast. Reconstr. Surg. 117: 1214, 2006.)
B
reast implant capsular contracture remains
one of the most common complications
for both aesthetic and reconstructive
breast surgery. Despite the importance of this
problem, the cause and treatment have remained unresolved for the past 40 years. Further
complicating this problem is that there are currently no reliable in vitro or in vivo models producing capsular contracture. Various animal
models have been reported in previous studies;
From the Department of Plastic Surgery, Nancy Lee and Perry
Bass Advanced Wound Healing Laboratory, University of
Texas Southwestern Medical Center.
Received for publication March 8, 2004; revised June 13,
2005.
Copyright ©2006 by the American Society of Plastic Surgeons
DOI: 10.1097/01.prs.0000208306.79104.18
1214
however, most lack the ability to produce the
pathologic state of contracture and, thus, correlation of proposed treatments for clinical capsular contracture are invalid in this setting.
Histologically, the human breast capsular
tissue is composed of an inner layer of fibrocytes and histiocytes, which is surrounded by a
thicker layer of collagen bundles arranged in a
parallel array.1,2 The outer layer is more vascular and is composed of loose connective tissue.
Although intuitively and clinically most would
consider the degree of capsule thickness to be
commensurate with the severity of capsular
contracture, this has never been definitively
proven, and some reports have found no correlation among contamination, thickness, and
clinical contracture.3
www.plasreconsurg.org
Volume 117, Number 4 • Model for Capsular Contracture
The literature is replete with earlier studies
that attempted to detect differences in capsule
characteristics between those formed around
smooth versus textured implants. Both gross andhistologic sections revealed a thicker capsule,
with increased cellularity surrounding the textured implants4,5; however, other reports have
produced contradictory results.6,7 Equally perplexing is the incongruity between studies with
animal models compared with human clinical
studies.4,5,8 Current data have yet to determine
the exact cause for contracture and thus no
completely effective prophylaxis or therapy has
been developed.5–9 Compounding the problem
is the use of various animal models for analysis of
capsular contracture when the animals themselves do not produce a pathologic capsular
state.6,10,11
Furthermore, a large body of conflicting data
exist on the mechanisms and various cell types
involved with the formation of the host capsular
contracture tissue response. As with any condition where the cause is unknown, there exists a
multitude of treatment modalities offered based
on anecdotal or clinically based experience. The
bulk of the literature on this subject is retrospective, unblinded, uncontrolled, and rarely uses
elegant scientific methodology.
The purpose of this study was to develop a
pathologic, reproducible, and reliable animal
model for capsular contracture that is similar to
human breast capsular contracture tissue. This
information can be used to help systematically
determine the cause of this problem and to allow
options for prevention and potential treatment
of capsular contracture.
Bristol, Tenn.) in 1 ml of 50 mM TrisCl (Sigma),
pH 7.4] into the implant pocket as a contractureinducing agent. The incision was closed in two
layers with subdermal 4-0 Vicryl (Ethicon, Inc.,
Somerville, N.J.) and 4-0 interrupted nylon suture.
Rabbits were killed at 2 or 8 weeks. Before the
animals were killed, each animal was anesthetized
and the dorsal back area was shaved. A small incision was made directly over the implant fill valve
through skin, panniculus carnosus, and capsule.
The incision traversing the capsule was sufficiently
small (⬍3 mm) to not impede the accurate assessment of intracapsular pressure. The Stryker
device was connected to the valve and opening
intracapsular pressure was recorded (Fig. 1). Subsequent pressures at 2-cc increments were recorded after equilibration as the implants were
overfilled. Representative capsule samples were
MATERIALS AND METHODS
Thirty-two New Zealand White rabbits underwent implantation with customized smooth saline
mini implants (30 cc; McGhan Medical, Santa Barbara, Calif.) under an approved institutional animal care protocol. Each implant was placed in the
subpanniculus carnosus plane in the dorsal back
region and filled to the manufacturer’s recommended 30-cc fill volume. One implant was placed
per rabbit, using sterile surgical technique.
The rabbits were divided into an experimental
(n ⫽ 16) and control subgroups (n ⫽ 16). The
experimental subgroup also underwent instillation of 5 cc of fibrin glue [fibrin glue is prepared
with 4 ml of rabbit cryo (Pel-Freez; Pel-Freez Biologicals, Rogers, Ark.), 500 ␮l of 10% CaCl (SigmaTau Pharmaceuticals, Gaithersburg, Md.), 1000
units of thrombin (Monarch Pharmaceuticals,
Fig. 1. (Above) Stryker pressure monitor setup next to the implanted mini implant. (Below) Stryker pressure monitor connected to the mini implant through a small capsular window.
1215
Plastic and Reconstructive Surgery • April 1, 2006
Fig. 2. The pressure-volume curve at 8 weeks; there was a significant increase in intracapsular pressure in the experimental group.
submitted in formalin for histologic evaluation for
tissue architecture and capsular thickness.
Human breast capsular tissue samples from
clinically normal breasts (implantation time, 6
months) and pathologically contracted capsule
(Baker III/IV; implantation time, 5 to 6 months)
were collected and processed using standard hematoxylin and eosin staining. The histologic sections were reviewed by a blinded pathologist and
the morphologic characteristics of the human capsule samples were characterized.
Statistics comparing the intracapsular pressures were performed using the two-tailed t test
demonstrating a significant difference between
the experimental and control groups. Statistical
significance is defined as p ⬍ 0.05.
RESULTS
The pressure-volume curve was generated at 2
and 8 weeks (Fig. 2). There was no significant
difference between the experimental and control
groups at 2 weeks; however, at 8 weeks there was
a significant increase in intracapsular pressure in
the experimental group. On gross examination of
the capsules, the control group capsule appeared
more transparent and had less vessel predominance on the capsular surface (Fig. 3, above). The
experimental group (Fig. 3, below) had a more
opacified capsule and in many cases appeared
thicker. The average capsular thickness (histologically measured) was 0.6 mm in the rabbit control
group, 1.0 mm in the rabbit experimental group
1216
and in human capsules, and 2.5 mm in human
capsule contractures. There was a non–statistically
significant increase in capsular thickness in the
experimental group.
Histology
Hematoxylin and eosin sections of rabbit control capsules at 8 weeks, rabbit contractures at 8
weeks, human capsules, and human contractures
were compared. Synovial-like reaction of fibrohistiocytic cells (synovial metaplasia) was most pronounced in the rabbit control capsule at 8 weeks,
focal in the rabbit contracture at 8 weeks, and
absent in the human contractures and control
capsules (which is not unexpected, as synovial
metaplasia is reported to be present in only 50
percent of cases).12
Inflammation (consisting of lymphocytes, histiocytes, and eosinophils) was moderate in the
8-week rabbit control capsule and mild in the
8-week rabbit contracture. The human capsule
demonstrated minimal inflammation, whereas the
human contracture showed mild inflammation.
The degree of fibrosis was greater in the 8-week
rabbit contracture and human contracture (Fig.
4) than in their counterparts (the 8-week rabbit
control and human capsules, respectively).
DISCUSSION
Capsular contracture is the most common
complication involving aesthetic and reconstructive breast surgery, with a reported incidence rang-
Volume 117, Number 4 • Model for Capsular Contracture
The true cause of capsular contracture remains elusive.18 Two prevailing theories have
emerged: the infectious hypothesis and the hypertrophic scar hypothesis. The infectious hypothesis,
which has been championed by Burkhardt and
supported by others,19 –23 implicates subclinical infection in the development of capsular contracture. Staphylococcus epidermidis, which is the most
common organism isolated from nipple secretions, is the most common organism cultured
from capsules excised during open capsulotomies.
Furthermore, acceleration of capsule formation
around silicone implants by addition of Staphylococcus aureus as an independent variable has been
reported.24
The hypertrophic scar hypothesis attempts to
implicate noninfectious stimuli, namely, hemato-
Fig. 3. (Above) Rabbit capsule at 8 weeks (control; the capsule is
transparent and has less vessel predominance on the capsular
surface). (Below) Rabbit capsule at 8 weeks (with fibrin glue); the
capsule is more opacified and thicker.
ing from 0.6 to 50 percent.13,14 An incidence of 8
to 15 percent15–17 may be cited as a more scientific
appraisal. Clinically, capsular contracture manifests on a continuum with varying degrees of severity, and is typically measured subjectively by
means of the Baker classification. Furthermore,
contracture may become clinically evident from
weeks to years after implantation.
Capsular contracture is the formation of fibrous scar tissue investing a foreign body or surgically implanted device. Artificial joints or heart
valves, central venous catheter ports, breast implants, and a multitude of additional surgical devices have been involved in the development of
capsule formation and its adverse consequences.
Capsule formation presumably plays a vital role in
the host’s response to a foreign body. Nevertheless, the results of this process may pose potential
serious health risks or adverse aesthetic sequelae.
Fig. 4. (Above) Experimental group rabbit contracture at 8
weeks (original magnification, ⫻40) showing areas of more
dense fibrous deposition in the mildly cellular mid zone; fibroblasts are widely separated by spindled fibroblasts. (Below) Human capsule contracture (original magnification, ⫻40) showing
hypocellular fibrous mid zone; central area shows dense collagen
without any fibroblasts; fibroblasts at periphery are widely separated by thick, dense bands of collagen fibers.
1217
Plastic and Reconstructive Surgery • April 1, 2006
mas, granulomas, or hereditary factors, which confer a foreign body reaction and resultant formation of a hypertrophic scar around an implanted
device. The underlying mechanism behind this
process involves the activation of the myofibroblast cells within the capsule, which supposed contractile elements exert the force necessary to produce capsular contracture. Myofibroblasts contain
the contractile elements actin and myosin and
have been identified inconsistently within the capsules of implanted devices; however, they have
proven difficult to culture and study in detail and,
when found in the capsule, are found in exceedingly small quantities, are located sporadically
throughout the capsule, and are not found to
attach to each other. This scenario poses an inconsistent model for the development of contractile forces necessary to produce contracture.
The purpose of this study was to consider a
novel pathologic animal model for capsular contracture. The fibrin glue inducing agent was discovered serendipitously in our laboratory; however, this places ample amounts of fibrinogen
around the implant, and the critical role of fibrinogen in capsule formation has been scientifically
established independent of our work.25 This agent
merely reliably produces conditions that are likely
to result in a contracted capsule.
Many different animal models for contracture
studies have been reported6,8,10,19,24 –28; however,
the majority consider the effect of a given therapy
on normal capsule formation.6,10,11,19,29,30 This minimizes and likely invalidates the significance/conclusions of many of these previous studies, as therapy needs to be directed at a pathologic capsule.
Other reports have used bacteria to stimulate the
formation of pathologic capsules; however, the
reproducibility and control of this model have not
been validated.19 It is our opinion that the cause
of contracture is multifactorial. In humans, there
exist capsular contracture–inciting agents that, for
known or unknown reasons, result in a contracture (i.e., hematoma, infection). The fibrin glue
inducing agent is no different. This agent simply
facilitates conditions that already are known to
produce capsule formation in a predictable fashion.
Furthermore, the correlation between animal
contracture and that of humans has not been substantiated. In fact, several studies using the rabbit
model have found contradictory results from our
clinical observation in humans.7,8 Most of these
studies have reported more pathologic capsules in
rabbits using textured implants,4,5 when it is generally accepted that textured implants produce
1218
less contracture in humans. The reason for this is
largely unknown; however, the use of a nonpathologic animal model is likely a major issue.
The histologic findings demonstrate a similar
increase in fibrosis in rabbit and human contracted capsule compared with respective controls. The differences in synovial metaplasia in the
specimens constitute a histologic detail that carries no clinicopathologic significance; however,
they were reported for the sake of completeness.
The end result is that the histologic analysis of the
rabbit contracture model is similar to human contracture.
We report for the first time, to the best of our
knowledge, a breast capsular contracture animal
model that mimics the histologic characteristics of
human breast capsular tissue. The degree of inflammation and fibrosis over time in the rabbit
contracture appears to correlate with those of the
human contracture, suggesting that the rabbit
capsule may be an optimal animal model for the
changes seen in human contractures. Despite
these findings, we acknowledge that the ultimate
model for the study of capsular contracture is the
human model, and all animal models, including
this one, will need to ultimately reconcile this fact.
CONCLUSIONS
Our model does produce pathologic and nonpathologic capsules histologically similar to the
human pathologic and nonpathologic capsule. Interestingly, our contracture-inducing agent (fibrin) has been implicated as a key player in the
formation of capsule formation in prior studies.28
Plastic surgeons have endured 40 years of darkness
in their true understanding of capsular contracture. We hope this model may not only provide a
platform for future investigation but allow us all to
see the “light” and provide insight into the true
cause of breast implant capsular contracture.
William P. Adams, Jr., M.D.
University of Texas Southwestern Medical Center at
Dallas
Southwestern Medical School
Department of Plastic Surgery
5323 Harry Hines Boulevard
Dallas, Texas 75390-9132
[email protected]
ACKNOWLEDGMENTS
The authors thank Debby Noble for excellent assistance with organizing much of this study. They also
thank Inamed Corporation for the manufacture and
donation of the specialized mini-implants.
Volume 117, Number 4 • Model for Capsular Contracture
REFERENCES
1. Kamel, M., Protzner, K., Fornasier, V., Peters, W., Smith, D.,
and Ibanez, D. The peri-implant breast capsule: An immunophenotypic study of capsules taken at explantation surgery. J. Biomed. Mater. Res. 58: 88, 2001.
2. Domanskis, E. J., Owsley, J. Q., Jr., et al. Histological investigation of the etiology of capsule contracture following augmentation mammaplasty. Plast. Reconstr. Surg. 58: 689, 1976.
3. Smahel, J. Histology of the capsules causing constrictive fibrosis around breast implants. Br. J. Plast. Surg. 30: 324, 1977.
4. Bern, S., Burd, A., May, J. W., Jr., et al. The biophysical and
histologic properties of capsules formed by smooth and textured silicone implants in the rabbit. Plast. Reconstr. Surg. 89:
1037, 1992.
5. Bucky, L. P., Ehrlich, H. P., Sohoni, S., et al. The capsule
quality of saline-filled smooth silicone, textured silicone, and
polyurethane implants in rabbits: A long-term study. Plast.
Reconstr. Surg. 93: 1123, 1994.
6. Clugston, P. A., Perry, L. C., Hammond, D. C., and Maxwell,
G. P. A rat model for capsular contracture: The effects of
surface texturing. Ann. Plast. Surg. 33: 595, 1994.
7. Coleman, D. J., Foo, I. T., and Sharpe, D. T. Textured or
smooth implants for breast augmentation? A prospective
controlled trial. Br. J. Plast. Surg. 44: 444, 1991.
8. Fagrell, D., Berggren, A., and Tarpila, E. Capsular contracture around saline-filled fine textured and smooth mammary
implants: A prospective 7.5-year follow-up. Plast. Reconstr.
Surg. 108: 2108, 2001.
9. Brohim, R. M., Foresman, P. A., Grant, G. M., Merickel, M.
B., and Rodeheaver, G. T. Capsular contraction around
smooth and textured implants. Ann. Plast. Surg. 30: 424, 1993.
10. Ajmal, N., Riordan, C. L., Cardwell, N., Nanney, L., and
Shack, R. B. Chemically assisted capsulectomy in the rabbit
model: A new approach. Plast. Reconstr. Surg. 112: 1449, 2003.
11. Caffee, H. H., and Rotatori, D. S. Intracapsular injection of
triamcinolone for prevention of contracture. Plast. Reconstr.
Surg. 92: 1073, 1993.
12. Rosen, P. P. Inflammatory and reactive tumors. In P. P. Rosen
(Ed.), Rosen’s Breast Pathology. Philadelphia: Lippincott Williams & Wilkins, 2001. Pp. 49–53.
13. Hakelius, L., and Ohlsen, L. Tendency to capsular contracture around smooth and textured gel-filled silicone mammary implants: A five year follow-up. Plast. Reconstr. Surg. 100:
1566, 1997.
14. Burkhardt, B., and Eades, E. The effects of Biocell texturing
and povidone-iodine irrigation on capsular contracture
around saline inflatable breast implants. Plast. Reconstr. Surg.
96: 1317, 1995.
15. Mentor Corp. Saline implant PMA. Available at: www.fda.
gov/cdrh/breastimplants/. Accessed October 1, 2000.
16. Inamed Corp. Saline implant PMA. Available at: www.fda.
gov/cdrh/breastimplants/. Accessed October 1, 2000.
17. Inamed Corp. Silicone Gel Implant PMA. Available at:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfAdvisory/details.cfm?mtg⫽388. Accessed June 1, 2003.
18. Rohrich, R. J., Kenkel, J. M., Adams, W. P., Jr., et al. Preventing capsular contracture in breast augmentation: In
search of the holy grail. Plast. Reconstr. Surg. 103: 1759, 1999.
19. Shah, Z., Lehman, J. A., and Tan, J. Does infection play a role
in breast capsular contracture? Plast. Reconstr. Surg. 68: 34,
1981.
20. Dobke, M. K., Svahn, J. K., Vastine, V. l., Landon, B. N., Stein,
P. C., and Parsons, C. L. Characterization of microbial presence at the surface of silicone mammary implants. Ann. Plast.
Surg. 34: 563, 1995.
21. Derman, G. H., Argenta, L. C., and Grabb, W. C. Delayed
extrusion of inflatable breast prostheses. Ann. Plast. Surg. 10:
154, 1983.
22. Virden, C. P., Dobke, M. K., Stein, P., Parsons, C. L., and
Frank, D. H. Subclinical infection of the silicone breast implant surface as a possible cause of capsular contracture.
Aesthetic Plast. Surg. 16: 173, 1992.
23. Schlenker, J. D., Bueno, R. A., Ricketson, G., and Lynch, J.
B. Loss of silicone implants after subcutaneous mastectomy
and reconstruction. Plast. Reconstr. Surg. 62: 853, 1978.
24. Kossovsky, N., Heggers, J. P., Parsons, R. W., and Robson, M.
C. Acceleration of capsule formation around silicone implants by infection in guinea pig model. Plast. Reconstr. Surg.
73: 91, 1984.
25. Chen, N. T., Butler, P. E. M., Hooper, D. C., and May, J. W.
Bacterial growth in saline implants: In vitro and in vivo studies. Ann. Plast. Surg. 6: 337, 1996.
26. Darouich, R. O., Meade, R., Mansouri, M. D., and Netscher,
D. T. In vivo efficacy of antimicrobe-impregnated salinefilled silicone implants. Plast. Reconstr. Surg. 109: 1352, 2002.
27. Ksander, G. A., Vistnes, L. M., and Fogarty, D. C. Experimental effects on surrounding fibrous capsule formation
from placing steroid in a silicone bag-gel prosthesis before
implantation. Plast. Reconstr. Surg. 62: 873, 1978.
28. Tang, L., Jennings, T. A., and Eaton, J. W. Mast cells mediate
acute inflammatory responses to implanted biomaterials.
Med. Sci. 95: 8841, 1998.
29. Raposo-do-Amaral, C. M., Tiziani, V., Trevisan, M. A., Pires,
C. H., and Palhare, F. B. Capsular contracture and silicone
gel: Experimental study. Aesthetic Plast. Surg. 16: 261, 1992.
30. Cherup, L. L., Antaki, J. F., Liang, M. D., and Hamas, R. S.
Measurement of capsular contracture: The conventional
breast implant and the Pittsburgh implant. Plast. Reconstr.
Surg. 84: 893, 1989.
1219
DISCUSSION
A Rabbit Model for Capsular Contracture: Development and
Clinical Implications
Boyd R. Burkhardt, M.D.
Tucson, Ariz.
I
have admired the work that Dr. Adams and his
team have published previously, and compliment them once again on their carefully controlled methodology and their nicely presented
results. Well-meaning colleagues often have constructive disagreements, however, and I do have
some with Dr. Adams. Readers will have to decide.
An accurate, reliable, and reproducible animal model for capsular contracture is indeed
needed. The authors do report an apparently
reliable and reproducible method for producing
measurable contracture around saline mini implants in rabbits. Whether the model is accurate
is discussed below. The experiment included a
control group, the measurement of contracture
was objective and reproducible, and the histology of the capsules was consistent with capsules
from around implants in humans. It is good
work, and the authors should be congratulated.
I do not believe, however, that this is an accurate model of the contracture process that occurs around breast implants in humans. For this
reason, I question the usefulness of this and
other similar animal research models in advancing our understanding and (it is hoped) preventing human contracture. This is a fundamental
dissent, and requires further explanation.
The authors note correctly that capsule formation is a normal physiologic response and that
our focus should be on capsular contracture
rather than on the capsule formation itself, a
distinction that has sometimes been ignored in
our literature and deserves emphasis. Although
acknowledging infection as one probable cause,
however, they believe the cause of contracture is
“multifactorial,” to include hematoma, granuloma, foreign body reaction, and hereditary factors, any one of which may theoretically stimulate an internal hypertrophic scar response that
then becomes a contracted capsule. This assumption is central to the potential usefulness of
their rabbit model: if contracture is just the final
common pathway for expression of a whole marReceived for publication October 4, 2005.
Copyright ©2006 by the American Society of Plastic Surgeons
DOI: 10.1097/01.prs.0000208309.47699.75
1220
ket basket of presumed nonbacterial causes, using fibrin glue to produce contracture in rabbits
and then developing treatments that modify this
tissue response is a rational approach. If the
presumed cause is limited to infection or bacterial contamination, however (and I confess some
personal bias here), I do not believe that working with glue-induced contracture in rabbits will
lead to remedies that are transferable to humans. If we examine published evidence, the
burden of proof falls clearly on those who assume that the cause is multifactorial and therefore reasonably duplicated by this rabbit response to fibrin glue.
Evidence for a bacterial etiology for human
contracture is abundant. Bacteria (mainly Staphylococcus epidermidis) have been cultured from 671
to 95 percent2 of contractures, 97 percent of
human breast milk samples,3 50 percent of intraoperative cultures of retromammary implant
pockets,4 and 50 percent of cultures of biopsy
specimens from uninfected breasts.5 Periprosthetic lactoceles, demonstrating a clear connection between the periprosthetic space and the
ductal system of the breast, are well documented
in our literature.6,7 To this I would add a personal observation that contractures in my own
practice often occur long after the initial surgery, following pregnancy and lactation. Bacterial contamination of mini inflatable implants
with S. epidermidis causes contracture in rabbits
that is reduced by intraluminal antibiotics.8,9
What is the evidence for other causes? An
“inherent” or genetic tendency is clearly inconsistent with a preponderance of unilateral contracture (do the right and left breasts really have
different genes or different tissue responses?).
Although previous authors have implicated inadequate initial dissection, 10 foreign body
reaction,11 hematoma,12 and the myofibroblast,13
the relevance of these studies, using the advantage of today’s knowledge, is quite thin.
If we are in fact dealing with a tissue response
to a securely buried, contaminated foreign body,
we must be especially cautious about attempts to
modify that tissue response. Intraluminal
steroids14 were a notable disaster that few of us
veterans would care to revisit. I do not pretend to
www.plasreconsurg.org
Volume 117, Number 4 • Discussion
have the answer, but I believe that if a rabbit
model must be used for research, a more appropriate model is that reported by Shah et al.,8,9
who used bacterial contamination to produce
contracture. I do question whether such studies
as the one at hand can lead to progress in the
prevention of human contracture, which I believe is the result of contamination from a
uniquely human environment of open, epithelium-lined, bacteria-filled breast ducts that simply
cannot (as yet) be duplicated on the backs of
rabbits.
Boyd R. Burkhardt, M.D.
4445 East Saranac Drive
Tucson, Ariz. 85718
[email protected]
REFERENCES
1. Burkhardt, B. R., Fried, M., Schnur, P. L., and Tofield, J. J.
Capsules, infection and intraluminal antibiotics. Plast. Reconstr. Surg. 68: 43, 1981.
2. Dubin, D. The etiology, pathophysiology, predictability, and
early detection of spherical scar contracture of the breast.
Presented at the 13th Annual Meeting of the American Society for Aesthetic Plastic Surgery, in Orlando, Fla., on May
19, 1980.
3. Boer, H. R., Guillermo, A., and MacDonald, N. Bacterial
colonization of human milk. South. Med. J. 74: 716, 1981.
4. Courtiss, E. H., Goldwyn, R. M., and Anastasi, G. W. The fate
of breast implants with infections around them. Plast. Reconstr. Surg. 63: 812, 1979.
5. Argenta, L. C., and Grabb, W. C. Studies on the endogenous
flora of the human breast and their surgical significance.
Presented at the Annual Meeting of the American Society of
Plastic and Reconstructive Surgeons, in New York, N.Y., on
October 20, 1981.
6. Hartley, J., and Schatten, W. Postoperative complications of
lactation after augmentation mammaplasty. Plast. Reconstr.
Surg. 47: 150, 1971.
7. Luhan, T. Giant galactoceles one month after bilateral augmentation mammaplasty, abdominoplasty and tubal ligation.
Aesthetic Plast. Surg. 3: 161, 1979.
8. Shah, Z., Lehman, J. A., and Tan, J. Does infection play a role
in breast capsular contracture? Plast. Reconstr. Surg. 68: 34,
1981.
9. Shah, Z., Lehman, J. A., and Stevenson, G. Capsular contracture around silicone implants: The role of intraluminal
antibiotics. Plast. Reconstr. Surg. 69: 809, 1982.
10. Cronin, T. D., Persoff, M. M., and Upson, J. Augmentation
mammaplasty: Complications and etiology. In J. Q. Owsley,
Jr., and R. A. Peterson (Eds.), Symposium on Aesthetic Surgery
of the Breast. St. Louis: Mosby, 1978, Pp. 272–282.
11. Vistnes, L. M., Ksander, G. A., and Kosek, J. Study of encapsulation of silicone rubber implants in animals: A foreign
body reaction. Plast. Reconstr. Surg. 62: 580, 1978.
12. Williams, C., Aston, S., and Rees, T. X. The effect of hematoma on the thickness of pseudosheaths around silicone
implants. Plast. Reconstr. Surg. 56:194, 1975.
13. Baker, J. L., Chandler, M. D., and LeVier, R. R. Occurrence
and activity of myofibroblasts in human capsular tissue surrounding mammary implants. Plast. Reconstr. Surg. 68: 905,
1981.
14. Oneal, R. M., and Argenta, L. C. Late side effects related to
inflatable breast prostheses containing soluble steroids. Plast.
Reconstr. Surg. 69: 641, 1982.
1221
Publication
BREAST
Long-Term Follow-Up of Breast Capsule
Contracture Rates in Cosmetic and
Reconstructive Cases
Marisa Marques, M.D.
Spencer A. Brown, Ph.D.
Isabel Oliveira, M.D.
M. Natália D. S. Cordeiro,
Ph.D.
Aliuska Morales-Helguera,
M.Sc.
Acácio Rodrigues, M.D.,
Ph.D.
José Amarante, M.D., Ph.D.
Porto, Portugal; and Dallas, Texas
Background: Silicone gel breast implants are associated with long-term adverse
events, including capsular contracture, with reported incidence rates as high as
50 percent. However, it is not clear how long the follow-up period should be and
whether there is any association with estrogen or menopausal status. In addition,
the placement of Baker grade II subjects in the majority of reports has been in
data sets of controls instead of capsular contracture.
Methods: A retrospective medical study (1998 to 2004) was performed in
women (n ⫽ 157) who received textured silicone breast implants for aesthetic
or reconstructive procedures at the Hospital of S. João (Portugal). Medical data
were collected that included the following: patient demographics, history, lifestyle factors, surgical procedures, and postoperative complications. Statistical
analyses included Pearson chi-square testing, logistic regression modeling, and
chi-squared automatic interaction detection (CHAID) methods.
Results: The reconstructive cohort had a great incidence of capsular contracture compared with the cosmetic cohort. If one considered no capsular contracture versus capsular contracture, the follow-up period should be longer than
42 months. However, if considering no capsular contracture and grade II subjects versus grade III or IV subjects, a longer follow-up period of 64 months was
determined. There was no association between capsular contracture and menopause/estrogen status.
Conclusions: Increased frequencies of capsular contracture were recorded in
breast reconstruction that were not attributable to estrogen or menopausal
status. On the basis of these results, the authors propose a follow-up period
longer than 42 months and the inclusion of Baker grade II subjects. (Plast.
Reconstr. Surg. 126: 769, 2010.)
S
ilicone gel breast implants for cosmetic augmentation and breast reconstruction have
been implanted worldwide since 1962.1 Multiple investigations have to date been alert for the potential adverse health effects of silicone breast
implants.2–12 Additional reports have focused on postoperative local complications and patient safety issues
in women receiving silicone breast implants.13–20
From the Departments of Plastic and Reconstructive Surgery
and Microbiology, Faculty of Medicine, and the REQUIMTE/
Department of Chemistry, Faculty of Sciences, University of
Porto; the Hospital de São João; and the Department of
Plastic Surgery Research, Nancy L. & Perry Bass Advanced
Wound Healing Laboratory, University of Texas Southwestern Medical School.
Received for publication September 10, 2007; accepted
March 11, 2010.
Copyright ©2010 by the American Society of Plastic Surgeons
DOI: 10.1097/PRS.0b013e3181e5f7bf
Capsular contracture is the most common and
severe complication associated with silicone breast
implants,13–20 despite innovations in shell surface
textures, implant shapes, inner gel composition,
surgical implantation techniques, and pocket
irrigation.21– 41 In cosmetic and reconstructive
breast surgery reports, the incidence of capsular
contracture ranged widely from 0 to 50 percent of
implantations.13–20,24,33,42– 46
The Baker classification system defines stages
of breast capsule clinical presentation into distinct
grades.46 Grade II is the first stage of capsular
contracture, and clinical interpretation of grade II
may be highly dependent on individual surgeons’
Disclosure: The authors have no financial interest
to declare in relation to the content of this article.
www.PRSJournal.com
769
Plastic and Reconstructive Surgery • September 2010
opinions. Although the clinical impact of grade II
is relevant to the continuum of breast capsule
formation, nevertheless, the majority of retrospective and prospective reports do not include grade
II subjects as breast capsule cases.47–50 The exclusion of grade II subjects in these reports may result
in underreporting of capsular contracture rates.
In this study, we report the occurrence and
severity of postoperative complications in a cohort
of Portuguese women who received silicone textured breast implants between 1998 and 2004.
Also, factors that might contribute to the development of capsular contracture rates (including
grade II subjects) were considered along temporal
trends with estrogens and menopausal status.
as they moved out of Porto or because no current
mailing address or phone numbers were available at
the time of the study. The reconstructive cohort was
composed of 88 patients with 115 breast implants
and with 27 patients having received bilateral breast
implants. The cosmetic cohort had 69 patients with
136 breast implants: 62 patients with 124 breast implants, two of whom had a tuberous breast deformity
and unilateral aplasia; and seven patients with 12
breast implants, with one woman having Poland syndrome. All cosmetic patients younger than 18 years
old (n ⫽ 4) had received implants following medical
indication, namely, severe asymmetry, aplasia of
breast tissue, or congenital malformation.
PATIENTS AND METHODS
Statistical Analysis
Postoperative local complications were analyzed independently for the entire study cohort
and individual clinical treatment cohorts and reported per woman and per implantation operation (SPSS, Inc., Chicago, Ill.). Possible associations among recorded data sets of patient
characteristics, surgical procedures, and complications were evaluated using Pearson chi-square
testing and logistic regression modeling.52 Trend
analysis was performed using the chi-squared automatic interaction detection (CHAID) method
(SPSS),53 using the likelihood ratio chi-square statistic as growing criteria, along with the Bonferroni 0.05 adjustment of probabilities, and setting
the minimum size for parent and child nodes at 10
and 5, respectively. Relative risks and 95 percent
confidence intervals were calculated for identified
characteristics of interest to examine strength and
precision of statistical associations.
CHAID has not been widely applied to trend
analyses in plastic surgery investigations, but
CHAID is one of the oldest tree-classification
methods originally proposed by Biggs et al.53 In
brief, CHAID is an exploratory method to examine relationships between a dependent variable
(e.g., capsular contracture) and a series of predictor variables (e.g., type of cohort, age at surgery, follow-up period) and their interactions. The
CHAID algorithm created adjustment cells by
splitting a data set progressively by means of a
classification tree structure where the most important predictor variables were chosen to maximize a chi-square criterion. The most significant
predictors defined the first split or the first branching of the tree. Progressive splits from the initial
variables resulted in smaller and smaller branches.
The result at the end of the tree-building process
is a series of groups that were different from one
Subjects and Data Collection
The study was approved by the Portuguese
Institutional Review Board for Human Subjects.
Existing medical records of women who had undergone breast implantation with customized textured silicone breast implants (McGhan Medical,
Santa Barbara, Calif.) between 1998 and 2004 in
the Hospital of S. João (Porto, Portugal) were
examined. A total of 224 women were identified,
with 104 women who had undergone cosmetic
breast augmentation (cosmetic cohort) and 120
women who had undergone postmastectomy reconstruction of the breast (reconstructive cohort).
From medical records, the following data were
collected: patient demographics, alcohol and medication use, medical history, surgical procedures, incision location, implant device placement,51 and
postoperative acute complications (hematoma, infection, or seroma). Postoperative chronic complication (capsular contracture, folds, wrinkles, breast
pain, and change of tactile sense) data were not
gathered from medical records. Self-reported complications related to satisfaction with implantation
surgery were collected using a self-administered
questionnaire. Women who answered the questionnaire were asked to attend a consultation to be further evaluated by the two trained plastic surgeons to
decrease subjectivity of this evaluation. The degree
of late capsular contracture was assigned by the plastic surgeons according to the Baker classification.46
Women from the initial cohort (157 of 224)
completed the self-questionnaire and attended the
consultation. The remaining 67 were then excluded
(n ⫽ 35 women, cosmetic cohort; n ⫽ 32, reconstructive cohort) to remove any potential bias that
might result from patients with incomplete data.
Women were excluded because of the loss of contact
770
Volume 126, Number 3 • Breast Capsule Contracture Rate
another on the dependent variable. Classification
trees lend themselves to be displayed graphically
and are far easier to interpret than numerical
interpretation from tables.
RESULTS
Baseline descriptive data for the cosmetic and
reconstructive patient cohorts are listed in Tables
1 and 2, respectively. Cosmetic patients were
younger at the time of surgery compared with
reconstructive patients (31.0 versus 48.6 years).
The average follow-up period was 35.4 months in
the cosmetic group compared with 48.5 months
in the reconstructive group. Contraceptive use was
reported by 56.5 percent of cosmetic patients,
whereas only 3.4 percent of reconstructive patients reported contraceptive use or hormone replacement therapy. Cosmetic patients also reported decreased use of psychotropic drugs (e.g.,
antidepressants, antianxiety, and hypnotics drugs)
compared with reconstructive patients (23.2 percent versus 52.3 percent, respectively). One
woman from each cohort (n ⫽ 2) had a connective
tissue disease (rheumatoid arthritis).
Among women in the cosmetic cohort, the
majority of silicone gel implants were placed subglandularly (84.1 percent), and the surgical approach was through the inframammary fold (59.4
percent). The majority of reconstructive patients
had not received radiotherapy (85.2 percent) or
tamoxifen (67.1 percent); chemotherapy was administered in 51.1 percent; the reconstructed
breast was the left side in 52.3 percent of the
patients, and 68.2 percent submitted to breast size
symmetrization.
Table 1. Baseline Characteristics for the
Cosmetic Cohort
Variable
No. of women with implants
(no. of breast implants)
Age at surgery, years
Mean
Range
Follow-up period, months
Mean
Range
Implant placement
Subpectoral
Subglandular
Dual-plane Tebbetts
Incision placement
Inferior periareolar
Axillary
Inframammary
Contraceptive drugs
No
Yes
No.
%
69 (136)
31.0
15⫺51
35.4
12⫺80
9
58
2
13.0
84.1
2.9
7
21
41
10.1
30.4
59.4
30
39
43.5
56.5
Table 2. Baseline Characteristics for the
Reconstructive Cohort
Variable
No. of women with implants
(no. of breast implants)
Age at surgery, years
Mean
Range
Follow-up period, months
Mean
Range
Symmetrizing breast
No
Breast implant
(with or without mastopexy)
Breast reduction
Bilateral breast reconstruction
Hormone therapy*
No
Yes
No.
%
88 (115)
48.6
25⫺73
48.5
12⫺96
28
31.8
22
33
5
25
37.5
5.7
85
3
96.6
3.4
*Including contraceptive drugs or hormone replacement therapy.
Acute Clinical Adverse Events
Acute complications were recorded in 20 reconstructive patients (8 percent) during the follow-up
period, with complications recorded as seroma
(8.0 percent), hematoma (4.5 percent), and perforation of the skin (3.2 percent) (data not shown).
Chronic Clinical Adverse Events
Chronic complication events were recorded
and are listed in Table 3. Overall, 81 percent (n ⫽
127) of all women had one or more postoperative
chronic events, ranging from less severe effects
(e.g., change in tactile sense) to complications
requiring additional surgical interventions, such
as severe capsular contracture. The distribution of
chronic complication frequency among women
was as follows: 23 percent of the patients had one
complication; 31 percent of the patients had two
complications; and 27 percent of the patients had
three or more complications. From a temporal
view of the clinical onset of chronic complications,
3 percent of the patients were diagnosed from 0 to
12 months postoperatively; 31 percent of the patients were diagnosed from 13 to 24 months; and
72 percent of the patients were diagnosed from 24
to 60 months.
The most frequent chronic adverse effect was
palpable implant folds (47.8 percent of all cases),
occurring in 42.0 percent of women from the cosmetic cohort and in 69.3 percent from the reconstructive cohort. Change of tactile sense also had
a high incidence (41.0 percent of all cases), with
89.8 percent in the reconstructive cohort reporting changes. Capsular contracture was the second
most common chronic complication, occurring in
771
Plastic and Reconstructive Surgery • September 2010
Table 3. Chronic Complications for Both Cohorts
Cosmetic
Cohort
(n ⴝ 69)
Chronic Complications
Capsular contracture
No
Unilateral
Bilateral
Palpable implant
folds
No
Unilateral
Bilateral
Visible skin wrinkles
No
Unilateral
Bilateral
Prolonged pain in
the breast
No
Unilateral
Bilateral
Change of tactile
sense
No
Unilateral
Bilateral
Reconstructive
Cohort
(n ⴝ 88)
No.
%
No.
%
57
9
3
82.6
13.0
4.4
46
41
1
52.3
46.5
1.2
40
12
17
58.0
17.4
24.6
27
48
13
30.7
54.5
14.8
59
7
3
85.5
10.1
4.4
72
14
2
81.8
15.9
2.3
59
4
6
85.5
5.8
8.7
78
9
1
88.7
10.2
1.1
61
4
4
88.4
5.8
5.8
9
67
12
10.2
76.1
13.7
*All reported cases were unilateral.
34.4 percent of all women and in 23.1 percent of
all implantations. Capsular contracture incidence
rates were significantly different between the cosmetic cohort (17.4 percent of women or 11.0 percent of implantations) and the reconstructive cohort (47.7 percent of women or 37.4 percent of
implantations; p ⬍ 0.05). Other chronic complications occurred less frequently (⬎10 percent of
all patients).
Furthermore, the occurrence of postoperative
complications had a marked influence on satisfaction index; for example, women without contracture were 1.6 times more likely to consider the
outcome either good or very good compared with
women with capsular contracture (relative risk,
1.6; 95 percent confidence interval, 1.2 to 2.2).
Capsular Contracture Characteristics
Baker capsular contracture grades for the cosmetic and reconstructive cohorts are listed in Table
4. As a percentage of patients, the reconstructive
cohort had 7.4- and 3.2-fold greater incidences of
Baker grade III and IV capsular contractures compared with the cosmetic cohort. When examined as
a function of clinical time when Baker grades were
assigned, 44 women (76 percent) of the 58 total
patients were diagnosed after 2 years after surgery. In
detail, five women (7 percent) from the cosmetic
cohort and 28 women (32 percent) from the recon-
772
Table 4. Capsular Contracture per Implant for
Both Cohorts
Grade* Cosmetic Cohort (%) Reconstructive Cohort (%)
I
II
III
IV
Total
121 (89.0)
5 (3.7)
2 (1.4)
8 (5.9)
136 (100)
72 (62.6)
9 (7.8)
12 (10.4)
22 (19.1)
115 (100)
*According to the Baker classification.
structive cohort developed capsular contracture
grade III/IV after the initial 2 years after implantation. Overall, the rate of grade III/IV capsular contracture per woman during the 8-year period of follow-up was 10.1 percent for patients undergoing
aesthetic surgery and 37.5 percent for breast reconstruction patients.
The occurrence of capsular contracture was
associated with the duration of follow-up and age
at the time of surgery (Table 5). Women with a
follow-up period longer than 42 months (relative
risk, 1.8; 95 percent confidence interval, 1.3 to 2.4)
or older women (relative risk, 3.6; 95 percent confidence interval, 1.6 to 7.9 for age 54 years or older
versus younger than 54 years) had increased incidences of capsular contracture (p ⬍ 0.001 for
both comparisons). Moreover, increased capsular
contracture occurred in the reconstruction group
of patients compared with the cosmetic cohort.
(relative risk, 1.7; 95 percent confidence interval,
1.4 to 2.3; p ⬍ 0.001). No associations between
capsular contracture cases and surgical procedures or other personal characteristics were
observed.
Using the CHAID decision tree (Fig. 1), the
type of cohort was identified as the determining
Table 5. Identified Variables Related to Capsular
Contracture for the Entire Cohort (n ⴝ 157)
Capsular
Contracture
(% of Women)
Variable
Follow-up period
ⱕ42 months
⬎42 months
Age at surgery
ⱕ54 years
⬎54 years
Hormone therapy*
No
Yes
Type of cohort
Reconstructive
Cosmetic
No
Yes
p
40.1
25.5
10.8
23.6
⬍0.001
60.5
5.1
24.8
9.6
⬍0.001
21.7
43.9
29.3
5.1
0.014
29.3
36.3
26.8
7.6
⬍0.001
*Including contraceptive drugs or hormone replacement therapy.
Volume 126, Number 3 • Breast Capsule Contracture Rate
Fig. 1. Prediction tree of capsular contracture by chi-square automatic interaction detection algorithm. The firstlevel split produced two initial branches: cosmetic and reconstructive. The type of cohort was identified as the
determining factor for developing capsular contracture, and the reconstructive group is predictive for positive
capsular contracture. The next splits indicated the best predictor variables for the reconstructive group, as the
follow-up period followed by the age at surgery. Within that group, a follow-up period of 42 months or less was
the best predictor for no capsular contracture and a follow-up of more than 42 months was predictive for positive
capsular contracture. For women with a follow-up of 42 months or less, capsular contracture was reported among
67.7 percent of women older than 54 years old compared with younger women (11.5 percent).
factor for developing capsular contracture. The
first-level split produced two initial branches: cosmetic (no capsular contracture; 82.6 percent) and
reconstructive (positive capsular contracture; 47.7
percent). The next splits indicated best predictor
variables for the cohort reconstructive group, as
the follow-up period followed by age at surgery.
Within that group, a follow-up period of 42
months or less was the best predictor for no capsular contracture (unadjusted, 74.3 percent) and
a follow-up of more than 42 months was predictive
for positive capsular contracture (unadjusted,
62.3 percent). For women with a follow-up of 42
months or less, capsular contracture was reported
among 67.7 percent of women older than 54 years
old compared with younger women (11.5 percent). The overall risk estimate according to the
classification tree was 0.240 (standard error of risk
estimate, 0.034), indicating that 75.8 percent of
the cases will be classified correctly by using the
decision algorithm based on the current tree. The
CHAID algorithm resulted in larger predictive values for occurrence of capsular contracture (72.2
percent) than logistic regression (57.4 percent).
A second CHAID decision tree analysis was
performed with grade II subjects placed in the no
capsular contracture group—similar to other re-
ports—versus grade III and IV subjects. The firstlevel split produced two initial branches: cosmetic
(no capsular contracture or grade II; 89.9 percent) and reconstructive (capsular contracture
grade III or IV; 37.5 percent). The next split indicated the best predictor variable for the reconstructive group, as the follow-up period. Within
that group, a follow-up period of 64 months or less
was the best predictor for no capsular contracture
or grade II (unadjusted, 73.4 percent), whereas a
follow-up of more than 64 months was predictive
for capsular contracture grade III or IV (unadjusted, 66.7 percent). The overall risk estimate
according to the classification tree was 0.255
(standard error of risk estimate, 0.035), indicating that 79.6 percent of the cases will be classified correctly by using the decision algorithm
based on the current tree.
Exogenous hormone use was reported in 56.5
percent of cosmetic patients (n ⫽ 39), with one
subject in menopause that used hormone therapy
replacement; of the remaining 68 women, 38 used
contraceptives (Fig. 2). Only 3.4 percent of reconstructive patients (n ⫽ 3) used hormone therapy.
Seventy-three patients were in menopause, with
two subjects who used hormone replacement ther-
773
Plastic and Reconstructive Surgery • September 2010
Table 6. Cross-Tabulation between Capsular
Contracture and Menopause According to Type
of Cohort
Capsular
Contracture
Menopause
Cosmetic cohort
Yes
No
Total
Reconstructive cohort
Yes
No
Total
Yes
No
Total
0
12
12
1
56
57
1
68
69
36
6
42
37
9
46
73
15
88
Table 7. Cross-Tabulation between Capsular
Contracture and Being Protected or Not by Estrogen
According to Type of Cohort
Fig. 2. Patients with or without menopause according to type
of cohort.
apy. Fifteen women were premenopausal, with
one who used contraceptives.
Subjects who were premenopausal or postmenopausal women using hormone therapy replacement were grouped and analyzed as
“estrogen protected” (Fig. 3). To clarify the relationships between menopause or women protected by estrogen with capsular contracture
rates according to type of cohort, two crosstabulations were performed (Tables 6 and 7).
Capsular
Contracture
Protected by Estrogen*
Cosmetic cohort
Yes
Total
Reconstructive cohort
Yes
No
Total
Yes
No
Total
12
12
57
57
69
69
35
7
42
36
10
46
71
17
88
*Including all women before menopause or in menopause with
hormone replacement therapy.
No associations between capsular contracture and menopause or estrogen status were
observed.
DISCUSSION
Fig. 3. Patients protected or not by estrogen according to
type of cohort.
774
Despite significant surgical efforts and precautions, capsular contracture continues to occur,23,29,31,54 –56 and the true cause of capsular contracture remains elusive.21,23,24,39 – 42,55–72 In our
report, the occurrence of local complications and
the frequency, severity, and long-term sequelae
were in the reported range as described in other
studies.13–20 Table 8 13–15,19,20,47–50,73–79 demonstrates
that reported capsular contracture rates vary
widely because of authors reporting various Baker
classification rates and follow-up time periods.
These data showed that the incidence of complications was elevated in reconstruction patients
compared with cosmetic augmentation patients.14,80
No acute complications occurred in the aesthetic
cohort, and all chronic complications were less prevalent in our study group. In our study, women with
breast implants for cosmetic reasons had a lower
body mass index than women who had undergone
Volume 126, Number 3 • Breast Capsule Contracture Rate
Table 8. Average Follow-Up versus Capsular Contracture
Study
Type of
Study
No. of Patients
Average
Follow-Up
11.5 mo
Spear et al., 200374
Prospective
85 cosmetic revisions
Adams et al., 200649
Prospective
235 (172 cosmetic
primary
augmentation; 63
reconstructive)
Henriksen et al.,
200580
Brown et al., 200519
Retrospective
Retrospective
2277
150 (118 cosmetic; 32
reconstructive)
Fruhstorfer et al.,
200413
Henriksen et al.,
200314
Cunningham et al.,
200747
Prospective
35
Prospective
1090
Prospective
14 mo
19.5 mo
21 mo
23 mo
955 (572 primary
augmentation; 123
revision-augmentation;
191 reconstruction;
69 revisionreconstruction)
Capsular Contracture
2% Baker grade II; no Baker
grade III–IV
Baker grade III–IV: 1.8% cosmetic
primary augmentation, 9.5%
reconstructive
4.3% (Baker grade II–IV)
Cosmetic, 2 cases; reconstructive,
3 cases; just Baker grade II; no
cases of Baker grade III–IV
0%
2 yr
4.1% (Baker grade II–IV)
2 yr
Baker grade III–IV: 0.8% primary
augmentation, 5.4 revisionaugmentation, 2.2% primary
reconstruction, 6% revisionreconstruction
Camirand et al.,
199975
Seify et al., 200576
Cunningham et al.,
200748
Prospective
Retrospective
Prospective
1007 (551 primary
augmentation; 146
revision-augmentation;
251 reconstruction;
59 revisionreconstruction)
3 yr
Baker grade III–IV: 8.1% primary
augmentation, 18.9 revisionaugmentation, 8.3% primary
reconstruction, 16.3% revisionreconstruction
Bengtson et al.,
200777
Prospective
3 yr
Baker grade III–IV: 5.9%
Spear et al., 200750
Prospective
6 yr
Baker grade III–IV: 14.8% primary
augmentation, 20.5% revisionaugmentation, 15.9% primary
reconstruction
Kjøller et al., 200173
Kulmala et al.,
200420
Retrospective
941 (492 cosmetic
primary
augmentation; 225
reconstructive; 224
revisions)
940 (455 cosmetic
primary
augmentation; 98
reconstructive; 162
revisions)
754
7 yr
11.4% of implantations
Hölmich et al.,
200778
Handel et al.,
200679
830
44
2.39 yr
34 mo
Retrospective
685
10.9 yr
Retrospective
190
19 yr
Retrospective
1529 (825 cosmetic;
264 reconstructive)
breast reconstruction, similar to previous studies10
that compared breast augmentation with breast
reduction and the general population.
In this study, 16 percent of capsular contractures (Baker grade III to IV) of the breast was
diagnosed after a 1.6-year period after initial
breast implantation. In the cosmetic study group,
no significant associations were formed between
surgical route or implant placement and any post-
23.3 yr
0%
20% (Baker grade II–IV)
17.7% (15.4% of implantations)
Baker grade II–IV
62%
Baker grade III–IV per 1000
patient-months: 1.99 cosmetic,
5.37 reconstructive, 4.36 revision
operative complication. Like Henriksen et al.,81 no
significant associations were observed between
body index mass, smoking habits, alcohol consumption, hormone therapy, and capsular contracture in our study groups.
Capsular contracture may be apparent within
the first year after implantation.14,15,33,81 However,
in our study, approximately 76 percent of cases of
capsular contracture (Baker grade II to IV) ap-
775
Plastic and Reconstructive Surgery • September 2010
peared just after 2 years; 10.1 percent and 37.5
percent of severe capsular contracture (Baker
grade III to IV) occurred in the aesthetic and
reconstructive cohorts, respectively, during the
8-year period of follow-up. Breiting et al.10 reported
an 18 percent rate of severe breast pain, indicative of
severe capsular contracture, and in a previous study
involving a subgroup of this population, they had
diagnosed a 45 percent rate of capsular contracture
(Baker grade II to IV) of the breast after a 5-year
period after breast implantation.82 Capsular contracture may also be symptomatic several years after
surgery.10,33,81,83
Using the CHAID decision tree, the determining factor for capsular contracture was the type of
cohort. The next splits indicated the best predictor variables for the cohort reconstructive level
being the follow-up period; if one considered no
capsular contracture versus capsular contracture,
the follow-up period should be longer than 3 years
6 months. However, if considering no capsular
contracture including grade II subjects versus
grade III or IV subjects, a longer follow-up period
of 5 years 4 months was determined. It is interesting that both CHAID tree decision analyses had
the same qualitative splits but with longer follow-up periods in grade III or IV subjects. This is
expected, as breast capsule formation is thought
to develop from grade II to grade III, and from
grade III to grade IV. These results underscore the
importance of considering grade II as an important clinical observation that should be included
in the capsular contracture analyses. Thus, we believe that a long follow-up period from grade II
and reconstructive patients should be considered
when studying local complications among women
receiving breast implants and other female agerelated factors such as menopause.
The protective role of estrogens in the progression of liver fibrosis84,85 and the fact that estrogen deprivation was being associated with declining dermal collagen content and impaired
wound healing is well known86; nevertheless, there
are no reports concerned with menopause or estrogens versus capsular contracture. The main
limitation of this study is the relatively small sample size and thus limited statistical power for observing relationships with rare outcomes, especially in the cosmetic cohort.
The authors for the first time report no association between capsular contracture and menopause or estrogen status. Therefore, the pathophysiology of capsule formation and subsequent
contracture developing metabolic pathways are
not estrogen derived. Our data suggest that grade
776
II subjects should be included in a capsule contracture analyses and a follow-up period longer
than 42 months should be considered. Our hope
is that the breast contracture “riddle” will be
solved in our lifetime so that our patients will not
have to confront recurrent and intractable capsular contractures.87
Marisa Marques, M.D.
Faculty of Medicine
University of Porto
Hospital de São João
Serviço de Cirurgia Plástica (piso 7)
Alameda Prof. Hernâni Monteiro
4202-451 Porto, Portugal
[email protected]
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Publication
Aesthetic Surgery Journal
http://aes.sagepub.com/
Effects of Fibrin, Thrombin, and Blood on Breast Capsule Formation in a Preclinical Model
Marisa Marques, Spencer A. Brown, Natália D. S. Cordeiro, Pedro Rodrigues-Pereira, M. Luís Cobrado, Aliuska Morales-Helguera,
Nuno Lima, André Luís, Mário Mendanha, Acácio Gonçalves-Rodrigues and José Amarante
Aesthetic Surgery Journal 2011 31: 302
DOI: 10.1177/1090820X11398351
The online version of this article can be found at:
http://aes.sagepub.com/content/31/3/302
Published by:
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On behalf of:
American Society for Aesthetic Plastic Surgery
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ERN
INT ATI
IBUTION
TR
AL CON
ON
Breast Surgery
Effects of Fibrin, Thrombin, and Blood on
Breast Capsule Formation in a Preclinical Model
Marisa Marques, MD; Spencer A. Brown, PhD; Natália D. S. Cordeiro,
PhD; Pedro Rodrigues-Pereira, MD; M. Luís Cobrado, MD; Aliuska
Morales-Helguera, PhD; Nuno Lima, MD; André Luís, MD; Mário
Mendanha, MD; Acácio Gonçalves-Rodrigues, MD, PhD; and José
Amarante, MD, PhD
Aesthetic Surgery Journal
31(3) 302­–309
© 2011 The American Society for
Aesthetic Plastic Surgery, Inc.
Reprints and permission:
http://www​.sagepub.com/
journalsPermissions.nav
DOI: 10.1177/1090820X11398351
www.aestheticsurgeryjournal.com
Abstract
Background: The root cause of capsular contracture (CC) associated with breast implants is unknown. Recent evidence points to the possible role of
fibrin and bacteria in CC formation.
Objectives: The authors sought to determine whether fibrin, thrombin, and blood modulated the histological and microbiological outcomes of breast
implant capsule formation in a rabbit model.
Methods: The authors carried out a case-control study to assess the influence of fibrin, thrombin, and blood on capsule wound healing in a rabbit
model. Eighteen New Zealand white rabbits received four tissue expanders. One expander acted as a control, whereas the other expander pockets
received one of the following: fibrin glue, rabbit blood, or thrombin sealant. Intracapsular pressure/volume curves were compared among the groups,
and histological and microbiological evaluations were performed (capsules, tissue expanders, rabbit skin, and air). The rabbits were euthanized at two or
four weeks.
Results: At four weeks, the fibrin and thrombin expanders demonstrated significantly decreased intracapsular pressure compared to the control group.
In the control and fibrin groups, mixed inflammation correlated with decreased intracapsular pressure, whereas mononuclear inflammation correlated
with increased intracapsular pressure. The predominant isolate in the capsules, tissue expanders, and rabbit skin was coagulase-negative staphylococci.
For fibrin and thrombin, both cultures that showed an organism other than staphylococci and cultures that were negative were associated with decreased
intracapsular pressure, whereas cultures positive for staphylococci were associated with increased intracapsular pressure.
Conclusions: Fibrin application during breast implantation may reduce rates of CC, but the presence of staphylococci is associated with increased
capsule pressure even in the presence of fibrin, so care should be taken to avoid bacterial contamination.
Keywords
capsule, tissue expander, fibrin, thrombin, blood, coagulase-negative staphylococci
Accepted for publication July 2, 2010.
Fibrosis is a major global health problem, but its cause,
pathogenesis, and diagnosis are not completely understood. Fibrosis may occur as a consequence of multiple
pathologic conditions, including keloids, Dupuytren contracture, postoperative adhesions, burns, postinfection
liver fibrosis, silica dust, asbestos, antibiotic bleomycin,
scleroderma, cardiac pacemakers, polypropylene meshes,
and—of special interest to aesthetic surgeons—breast
implant capsular contracture (CC).1-3
The cause of CC remains largely undetermined, with clinically-reported incidences ranging from 8% to 45%.4-9 Prior
investigations of CC have focused on microorganisms found
in the capsule or outer implant surface,10-22 on inflammatory
responses,1,23,24 and on histological characteristics of the capsule.25-32 Two reports found correlations between CC and
hematoma.32,33
Histologically, human CC tissue comprises an inner layer
of fibrocytes and histiocytes, surrounded by a thicker layer
Dr. Marques is in the Department of Plastic and Reconstructive Surgery,
Faculty of Medicine, University of Oporto, Hospital of São João,
Portugal. Dr. Cordeiro and Dr. Morales-Helguera are in the Department
of Chemistry, Faculty of Sciences, University of Oporto, Portugal. Dr.
Rodrigues-Pereira is in the Department of Pathology, Faculty of Medicine,
University of Oporto, Hospital of São João, Portugal. Dr. Cobrado is
in the Department of Microbiology, Faculty of Medicine, University
of Oporto, Portugal. Dr. Lima, Dr. Luís, and Dr. Mendanha are in the
Department of Experimental Surgery, Faculty of Medicine, University
of Oporto, Portugal. Dr. Gonçalves-Rodrigues is the Department Head
of Microbiology, Faculty of Medicine, University of Oporto, Portugal.
Dr. Amarante is the Department Head of Surgery, Faculty of Medicine,
University of Oporto and was the Department Head of Plastic and
Reconstructive Surgery, Hospital of São João, Portugal.
Corresponding Author:
Dr. Marisa Marques, Hospital de São João, Serviço de Cirurgia
Plástica, Alameda Prof. Hernâni Monteiro, 4202 Porto, Portugal.
E-mail: [email protected]
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Marques et al
303
of collagen bundles arranged in a parallel array.34,35 The
outer layer is more vascular and contains loose connective
tissue. From a clinical perspective, authors seems to agree
that the degree of capsule thickness is proportionate to the
severity of the CC, but this has never been definitively
proven and some reports found no correlations among
microbial contamination, thickness, and CC.36 However, we
do know that transforming growth factor beta 1 (TGF-β1),
connective tissue growth factor, osteopontin, interleukin-4
(IL-4), IL-6, IL-10, IL-13, IL-21, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor 1,
platelet-derived growth factor, oncostatin M, and endothelin
1 (see Sticherling37) all promote fibrosis.1
Numerous reports have associated fibrin glue with
improved parameters of wound healing38-41 and reduced
quantity and consistency of adhesions, even in the case of
polypropylene meshes.2,3 To the best of our knowledge, only
one study focused on the impact of autologous fibrin glue on
capsule formation as a contracture-inducing agent, and no
reports exist for commercially-available fibrin products.26 In
preclinical reports, several molecules were found to reduce
CC27,42,43; nevertheless, these compounds are not currently
available in clinical practice. However, the fibrin-containing
commercial products are widely used clinically and are an
attractive adjunct for patients receiving breast implants.
The purpose of this study was to perform a comprehensive evaluation (based on a rabbit model26) of the relationships among intracapsular pressure, histological characteristics,
and infection surrounding the tissue expander in the capsule,
in the rabbit skin, and in the operating room air. To clarify
whether hematoma is associated with CC, the study was
conducted with tissue expanders surrounded by rabbit blood
to simulate a hematoma, as well as with tissue expanders in
the presence of thrombin (FloSeal, Baxter US, Deerfield,
Illinois), an absorbable hemostatic agent that contains no
fibrinogen and requires contact with blood for the clot to be
activated. To study the implications of wound healing in
development of CC, the implant pocket was instilled with
fibrin (Tissucol/Tisseel, Baxter US), a hemostatic agent with
adhesive properties.
Methods
Eighteen New Zealand white female rabbits (3-4 kg) were
implanted with textured saline tissue expanders (20 mL,
Allergan, Santa Barbara, California) with intact connecting
tubes and ports, in accordance with an approved institutional animal care protocol. Before surgery, the animals
were washed with Betadine surgical scrub (Purdue Pharma
LP, Stamford, Connecticut), which contains 7.5% povidoneiodine, and their skin disinfected with Betadine solution,
which contains 10% povidone-iodine. The surgical procedure
was performed in a veterinary operating room with aseptic
techniques. Penicillin G (40,000 U/kg) was immediately
administered intramuscularly to the subjects was intraoperatively. Talc-free gloves were worn at all times during
the procedure. Pockets were atraumatically dissected
under direct vision in the subpanniculus carnosus along
the back region of each rabbit. Attention was paid to
hemostasis and blunt instrumentation was avoided; there
was no obvious bleeding. A new pair of talc-free gloves
was placed on the surgeon’s hands before tissue expander
insertion, with minimal skin contact.
One control and three experimental tissue expanders
were placed in each rabbit. The experimental expanders
received one of the following: 1 mL of fibrin glue spray
(Tisseel/Tissucol), 2 mL of rabbit blood to simulate a
hematoma, or 5 mL of thrombin sealant (FloSeal) in the
expander pocket. A pressure-measuring device (Stryker
Instruments, Kalamazoo, Michigan) was connected to
each tissue expander port. Intraexpander pressure was
recorded immediately before filling and in 5-mL increments until the tissue expanders were overfilled. Each
tissue expander was filled to 20 mL.
The rabbits were euthanized at two or four weeks.
Beforehand, each animal was anesthetized and the dorsal back
area was shaved. The pressure monitor was connected again
to the tissue expander port, and intracapsular pressures were
recorded in 5-mL increments as the expander was drained,
before any incision in the capsule. Capsule samples were submitted for histological and microbiological evaluation.
Microbiological Assessments
Air. Operating room air samples (n = 36) were collected
during all procedures with the MAS 100-Eco air sampler (EMD
Chemicals, Inc., Gibbstown, New Jersey) at a flow rate of 100
L per minute. Identification of bacterial and fungal isolates followed standard microbiological procedures. Gram-positive
cocci were characterized by biochemical methods. Catalasepositive and coagulase-positive colonies were identified as
Staphylococcus aureus; catalase-positive and coagulase-negative colonies were identified as coagulase-negative
staphylococci. Gram-negative bacilli were characterized with
Vitek 2 software (VT2-R04.02, bioMérieux, Inc., Durham,
North Carolina). Fungi were characterized following their
macroscopic appearance and microscopic morphology.
Rabbit skin. A total of 54 contact plates were pressed to
shaved dorsal skin surfaces (18 brain-heart agar, 18 mannitol salt agar, and 18 Sabouraud agar). The brain-heart
and mannitol salt contact plates were incubated for three
days at 28°C; the Sabouraud contact plates were incubated for seven days at 28°C. Bacterial and fungal colonies
were counted and reported as colony-forming units per
square centimeter. For the identification of the bacteria
and fungi grown, the same methods listed above were
applied.
Capsules and tissue expanders. Excised implants and representative capsule samples were incubated at 37°C for
three days in brain-heart agar plates and examined daily;
changes in turbidity of the broth media were considered
positive and were subcultured
in solid agar media.
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304
Aesthetic Surgery Journal 31(3)
Characterization of microbial isolates followed the abovedescribed procedures.
Histological Assessment
Capsule specimens were fixed with 10% buffered formalin
and embedded in paraffin. Sections were stained with
hematoxylin and eosin and histologically evaluated for tissue inflammation and capsular thickness. The type and
intensity of the inflammatory infiltrate were analyzed.
Inflammation was grouped into three categories: mononuclear/chronic (lymphocytes, plasmocytes, and histiocytes),
mixed/subacute (mononuclear cells and eosinophils), and
polymorph/acute (eosinophils and heterophils). Inflammatory infiltrate intensity was categorized according to the
following criteria: absent (−), mild (+), moderate (++),
and severe (+++).25
Samples were stained with Masson trichrome44 to characterize the collagen (loose, slightly dense [≤ 25%] or
more dense [> 25%]), the organization of collagen fibers
(parallel or haphazard), the angiogenesis (absent, mild,
moderate, or high), and the fibroblast density (mild, moderate, or high). Histological sections were reviewed and
graded by a pathologist blinded to the protocol.
Statistical Analysis
Data were grouped according to the type of product applied
to the tissue expander: none (control), blood (blood),
Tissucol/Tisseel (fibrin), and FloSeal (thrombin). Each was
analyzed for the rabbits euthanized at two and four weeks
after surgery, as well as for all 18 rabbits. One-way analysis
of variance was applied to compare the intraexpander pressure before insertion. A two-tailed paired t-test and the
nonparametric alternative Wilcoxon signed rank test were
applied to determine whether continuous variables (intracapsular pressure and histologically measured thickness)
were significantly different between the control and experimental groups. Categorical variables were evaluated by chisquare statistics and by phi, Cramer V, and contingency
coefficients. Statistical significance was presumed at p ≤ .05.
Major trends within each group were further examined by
the chi-square automatic interaction detection (CHAID)
method,45 based on the likelihood ratio chi-square statistic
as growing criteria, along with a Bonferroni 0.05 adjustment
of probabilities. All analyses were carried out with SPSS
version 16 (SPSS, Inc., Chicago, Illinois).
Results
Intracapsular Pressure
No significant differences were observed in the pressurevolume curves between the control and experimental groups
at baseline (tissue expander introduction) or at two weeks.
At four weeks, rupture was observed during pressure
Figure 1. Pressure-volume curves at four weeks. There
was a significant difference in intracapsular pressure
in the thrombin (FloSeal) and fibrin (Tissucol/Tisseel)
experimental groups.
measurement with six capsules in the control group, five
capsules in the blood group, and one capsule in thrombin
group; no capsule ruptures in the fibrin group were noted. To
avoid reducing the sample size, the ruptured capsules were
not excluded from statistical analyses, but it is important to
note that the pressure levels measured before capsule rupture
were maintained after additional saline was added. At four
weeks, significantly decreased intracapsular pressures were
registered in the fibrin group (p ≤ .0006) and thrombin group
(p ≤ .003) (Figure 1).
Histology
The average capsular thicknesses were similar among all
groups at two and four weeks (Table 1). At two weeks,
mixed types of inflammatory cells were predominant in the
capsules, and no statistically significant differences were
found among the groups. At four weeks, mononuclear cells
were predominant in the control, blood, and thrombin
groups; in the fibrin group, mixed cells were predominant.
However, these differences were not statistically significant.
At both two and four weeks, trends in the intensity of
inflammation showed no significant difference (Table 2).
Fibrosis developed in all capsules at two and four
weeks. No significant differences were observed regarding
the organization of collagen fibers between the control and
experimental groups. At two weeks, more dense collagen
(> 25%) was found in the control group, whereas loose
and slightly dense collagen (≤ 25%) was found in the
blood group (p = .023). At both two and four weeks,
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Marques et al
305
more than one isolate in 10% (seven); tissue expanders
yielded a single isolate in 32% (23) and more than one
isolate in 15% (15). No fungi were recovered from the
removed capsules or tissue expanders in any rabbits.
Similar bacterial isolates were cultured from rabbit skin.
The predominant isolated bacteria was coagulase-negative
staphylococci, in 16 of 18 euthanized rabbits (89%). A single
skin sample was culture-negative, whereas two samples
yielded more than one bacteria. Isolated bacteria from rabbit
skin were not different from those removed from the capsules
and tissue expanders. Coagulase-negative staphylococci were
also isolated from all air samples. Other isolates included
gram-positive bacilli and Staphylococcus aureus (although
these were found much less frequently). Several species,
such as Penicillium spp., Aspergillus niger, and zygomycetes,
were recovered from the operating room air.
Statistical analyses revealed no significant differences
in the frequency of culture positivity and the type of bacteria among all the groups and no significant correlation
between the microbial presence and the histological characteristics.
Table 1. Average Capsular Thickness of Control Versus Experimental
Groups
Group
Two Weeks, mm
Four Weeks, mm
Control
0.83 ± 0.085
0.64 ± 0.078
Blood
1.02 ± 0.207
0.78 ± 0.572
Fibrin: Tissucol/Tisseel
0.89 ± 0.082
0.72 ± 0.083
Thrombin: FloSeal
0.90 ± 0.064
0.71 ± 0.105
increased angiogenesis was observed in the control group
(moderate/ high) versus the blood group (negative/mild)
(p = .018). At four weeks, significant differences were
found in the fibroblast density between the control and
blood groups (p = .047)—mild in the control group and
moderate in the blood group.
Microbiology
CHAID Modeling Associations
At two and four weeks, bacteria were isolated in 53% of
the capsules (38 of 72) and 47% of the tissue expanders
(34 of 72). The specimens included coagulase-negative
staphylococci (41%), Escherichia coli (10%), Staphylococcus
aureus (8%), Pseudomonas spp (0.7%), and other gramnegative bacilli (0.7%). In the capsules, the predominant
isolated bacteria was coagulase-negative staphylococci,
identified in 53% at two weeks (19 of 36), decreasing to
33% at four weeks (12 of 36). In tissue expanders, coagulase-negative staphylococci was identified in 44% at two
weeks (16 of 36), decreasing to 22% at four weeks (eight
of 36). Capsules yielded a single isolate in 43% (31) and
At four weeks, statistical analysis with CHAID modeling
demonstrated association with intracapsular pressure at 20
mL for the control and fibrin groups. The determining factor
for intracapsular pressure at four weeks was the type of
inflammatory cells (Figure 2). The CHAID analysis showed
in both trees that mixed inflammation was related to
decreased intracapsular pressure and that mononuclear
inflammation was related to increased intracapsular pressure. In the control tree, moderate inflammation was related
to decreased pressure in the capsules with mononuclear
Table 2. Outcomes for Capsule Inflammation of Control Versus Experimental Groups
Group
Type of Inflammatory Cells
Two Weeks, %
Four Weeks, %
Intensity
Two Weeks, %
Four Weeks, %
Control
Mononuclear: chronic
22.2
55.6
Mild
11.1
55.6
0.0
0.0
Moderate
77.8
44.4
Mixed: active chronic
77.8
44.4
High
11.1
0.0
Mononuclear: chronic
33.3
55.6
Mild
33.3
33.3
0.0
0.0
Moderate
66.7
66.7
Mixed: active chronic
66.7
44.4
High
0.0
0.0
Mononuclear: chronic
11.1
22.2
Mild
0.0
22.2
0.0
0.0
Moderate
66.7
33.3
Mixed: active chronic
88.9
77.8
High
33.3
44.4
Mononuclear: chronic
22.2
77.8
Mild
11.1
66.7
0.0
0.0
Moderate
77.8
33.3
77.8
22.2
High
11.1
0.0
Polymorph: acute
Blood
Polymorph: acute
Fibrin: Tissucol/Tisseel
Polymorph: acute
Thrombin: FloSeal
Polymorph: acute
Mixed: active chronic
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306
Aesthetic Surgery Journal 31(3)
A
PRESSURE
≤ 81: 5 (55.6%)
>81: 4 (44.4%)
TYPE OF INFLAMMATORY CELLS p < 0.0007
MIXED
≤ 81: 4 (100%)
>81: 0 (0%)
MONONUCLEAR
≤ 81: 1 (20%)
>81: 4 (80%)
INTENSITY p < 0.025
MILD
≤ 81: 0 (0%)
>81: 4 (100%)
B
MODERATE
≤ 81: 1 (100%)
>81: 0 (0%)
PRESSURE
< 70: 6 (66.7%)
≥ 70: 3 (33.3%)
TYPE OF INFLAMMATORY CELLS p < 0.017
MIXED
< 70: 6 (85.7%)
≥ 70: 1 (14.3%)
MONONUCLEAR
< 70: 0 (0%)
≥ 70: 2 (100%)
Figure 2. Decision tree by CHAID algorithm for histology
data at four weeks. (A) control group; (B) experimental
fibrin group (Tissucol/Tisseel).
inflammatory cells, whereas capsules with mild inflammation had increased pressure.
CHAID classification analyses with intracapsular pressure
at 20 mL for the fibrin and thrombin groups showed that the
determining factor for intracapsular pressure at four weeks
was the kind of bacteria isolated from tissue expanders
(Figure 3). Cultures showing an organism other than
Staphylococcus (E. coli, Pseudomonas spp) and negative cultures (those with no contamination) were correlated with
decreased intracapsular pressure. Coagulase-negative staphylococci and Staphylococcus aureus were correlated with
increased intracapsular pressure.
Discussion
The major findings of our study were observed on capsules
and tissue expanders in the rabbits euthanized at four weeks.
Compared to the control group, the fibrin and thrombin
groups showed significantly decreased intracapsular pressures. The fibrin group was the only group with no capsular
ruptures during pressure measurement. For the control and
fibrin groups, mixed inflammation was associated with
decreased intracapsular pressures, whereas mononuclear
inflammation was associated with increased intracapsular
pressures. For the fibrin and thrombin groups, cultures with
bacteria other than staphylococci and negative cultures were
associated with decreased intracapsular pressure, whereas
staphylococci cultures were associated with increased intracapsular pressure. In the blood group, increased fibroblast
densities were observed as compared to the control group.
Increased angiogenesis was observed in the control group
compared to the blood group. Average capsular thicknesses,
the type and intensity of the inflammatory infiltrate, and collagen density and organization were similar among all groups.
Also, the isolated bacteria in capsules, tissue expanders, and
rabbit skin were similar among the groups. In capsules, tissue
expanders, and rabbit skin, the predominant isolated bacteria
was coagulase-negative staphylococci, which was also isolated from all air samples. No fungi were recovered from
capsules, tissue expanders, or rabbit skin, but they were isolated from all air samples.
Of note, this study was performed with tissue expanders to measure the capsule pressure directly46 to achieve
more accurate results. Similar capsules and increased
pressure levels were observed in both the control group
and the blood group. On the basis of wound-healing principles,47 we can conclude that increased pressure levels
and capsule rupture rates correlate with contracture. That
increased angiogenesis is associated with fibrosis has been
documented,31,46,48 supporting the major trends observed
in CC development in the control group of this study.
FloSeal requires blood for activity; therefore, given the
thrombin group results, we may conclude that an active
hemostasis is indispensable to preventing CC, although it is
unnecessary with a hemostatic commercial product.
However, the fibrin group demonstrated mixed inflammation, which correlated with decreased intracapsular pressures as compared with the control group. This is consistent
with other reports observing that the activation of fibrosis
in the early implant period may be the major mechanism
for CC development.25
In our study, inflammation was not significantly correlated with capsular thickness, which is consistent with the
results reported by Siggelkow et al.25 Sead et al studied
fibrin sealant prepared from a Tisseel kit without aprotinin
and observed a reduction in the extracellular matrix and
TGF-β1, especially from adhesion fibroblasts, which may
indicate a role in the reduction of postoperative adhesion
development.49 It is well known that fibrosis is associated
with excessive collagen extracellular matrix formation, cell
proliferation, and activation of myofibroblasts. In this context, macrophages and mast cells have been implicated as
important participants in the inflammatory process involving fibrosis.1 Macrophages contribute to this process by the
production of TGF-β1 and IL-6.50
In a study by Ruiz-de-Erenchun et al,51 TGF-β1 inhibitor
peptide applied in a matrix with tetraglycerol dipalmitate
was significantly effective in achieving a reduction in
periprosthetic fibrosis after placement of silicone implants.
Interestingly, in our fibrin group, mixed inflammation was
correlated with decreased intracapsular pressure, but intracapsular pressure increased in the presence of Staphylococcus
infection. Our results suggest that fibrin plays a role in preventing CC, that the bacterial colonization of mammary
implants may be partially responsible for CC, and that
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Marques et al
A
307
PRESSURE
< 70: 6 (66.7%)
≥70: 3 (33.3%)
MICROBIOLOGY p < 0.001
Other than staphylococci and
No contaminated
< 70: 6 (100%)
≥70: 0 (0%)
B
Staphylococci
< 70: 0 (0%)
≥70: 3 (100%)
PRESSURE
<64: 5 (55.6%)
≥64: 4 (44.4%)
MICROBIOLOGY p < 0.001
Other than staphylococci and
No contaminated
<64: 5 (83.3%)
≥64: 1 (16.7%)
Staphylococci
<64: 0 (0%)
≥64: 3 (100%)
Figure 3. Classification tree by CHAID algorithm for
microbiology data at four weeks. (A) experimental fibrin
group (Tissucol/Tisseel); (B) experimental thrombin group
(FloSeal). Bacteria other than Staphylococcus (Escherichia
coli, Pseudomonas spp) and those with no contamination
were considered negative cultures; Staphylococcus
contamination included coagulase-negative staphylococci or
Staphylococcus aureus.
coagulase-negative staphylococci may play a large role.52-66
As reported in the literature, infection of implanted medical
devices is commonly mediated by formation of bacterial
biofilms.67-70 However, Pajkos et al13 reported that biofilm
was found with scanning electron microscopy in a single
culture-negative sample. It is interesting that extensive
amorphous biological deposits were observed with scanning electron microscopy, even in the absence of bacterial
structures. Moreover, because of the low pathogenicity of
coagulase-negative staphylococci and the existence of
microorganisms in a dormant phase within the biofilm
around the implant, CC does not usually clinically manifest
until some remote time after placement of mammary
implants.13,67-70 For all of these reasons, we did not consider
biofilm investigation in this preclinical study. All the methods for biofilm investigation are expensive, not routinely
used, and require a longer follow-up period.
We euthanized the rabbits at two or four weeks to
study capsule formation and wound healing.47 Our study
demonstrated wound-healing results at two weeks among
all groups that were similar to those from a report by
Adams et al.26 Our results differed in that intracapsular
pressure decreased with fibrin glue application, whereas
their results showed increased pressure.26 This may be
explained in part by the fact that our data were collected
at four weeks, whereas that study focused on capsules at
eight weeks. Also, the Adams et al study utilized an
autologous fibrin glue of unknown fibrin concentration,
whereas our study utilized a commercial fibrin product
widely studied and used in clinical practice (Tissucol/
Tisseel) to reduce polypropylene mesh adhesions.2,3 To the
best of our knowledge, this is the first report examining CC
with a commercially-available fibrin product. In addition,
this study is the first to include an examination of bacterial
contamination from rabbit skin and operating room air.
Furthermore, our study (which is an extension of the
Adams study) placed expanders in New Zealand white
rabbits rather than mice, given that the rabbits had the
capacity to support all four expanders. The data in porcine
models are limited.
One limitation of this study was the use of tissue expanders instead of commercial silicone breast implants, which are
not available in an appropriate size for the rabbit model. One
strength of our study was the statistical analyses among the
four groups with the CHAID method, a sophisticated algorithm widely used in other disciplines because it models a
single variable among multiple variables. Future studies may
expand upon our results by extending the follow-up period,
by inserting breast implants, instead of tissue expanders,
sprayed with fibrin, or by focusing on fibrosis that may influence or modulate CC.
Conclusions
Fibrin applied in the breast implant pocket may reduce
CC. With its relatively-well-documented safety profile,
fibrin-containing compounds can be considered an attractive adjunct in breast implant surgeries. Clinical strategies
for preventing bacterial contamination during surgery are
crucial, given that Staphylococcus (mainly, coagulasenegative staphylococci) may promote CC even with fibrin.
Disclosures
The authors declared no conflicts of interest with respect to
the authorship and publication of this article.
Funding
The authors received no financial support for the research and
authorship of this article.
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Publication
Aesthetic Surgery Journal
http://aes.sagepub.com/
Effects of Coagulase-Negative Staphylococci and Fibrin on Breast Capsule Formation in a Rabbit Model
Marisa Marques, Spencer A. Brown, Natália D. S. Cordeiro, Pedro Rodrigues-Pereira, M. Luís Cobrado, Aliuska Morales-Helguera,
Lara Queirós, André Luís, Rui Freitas, Acácio Gonçalves-Rodrigues and José Amarante
Aesthetic Surgery Journal 2011 31: 420
DOI: 10.1177/1090820X11404400
The online version of this article can be found at:
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ERN
INT ATI
IBUTION
TR
AL CON
ON
Research
Effects of Coagulase-Negative Staphylococci and
Fibrin on Breast Capsule Formation in a Rabbit
Model
Aesthetic Surgery Journal
31(4) 420­–428
© 2011 The American Society for
Aesthetic Plastic Surgery, Inc.
Reprints and permission:
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DOI: 10.1177/1090820X11404400
www.aestheticsurgeryjournal.com
Marisa Marques, MD; Spencer A. Brown, PhD; Natália D. S. Cordeiro, PhD;
Pedro Rodrigues-Pereira, MD; M. Luís Cobrado, MD; Aliuska MoralesHelguera, PhD; Lara Queirós, MD; André Luís, MD; Rui Freitas, MD; Acácio
Gonçalves-Rodrigues, MD, PhD; and José Amarante, MD, PhD
Abstract
Background: The etiology and ideal clinical treatment of capsular contracture (CC) remain unresolved. Bacteria, especially coagulase-negative
staphylococci, have been previously shown to accelerate the onset of CC. The role of fibrin in capsule formation has also been controversial.
Objective: The authors investigate whether fibrin and coagulase-negative staphylococci (CoNS) modulate the histological, microbiological, and clinical
outcomes of breast implant capsule formation in a rabbit model and evaluate contamination during the surgical procedure.
Methods: Thirty-one New Zealand white female rabbits were each implanted with one tissue expander and two breast implants. The rabbits received (1)
untreated implants and expanders (control; n = 10), (2) two implants sprayed with 2 mL of fibrin and one expander sprayed with 0.5 mL of fibrin (fibrin; n = 11),
or (3) two implants inoculated with 100 µL of a CoNS suspension (108CFU/mL—0.5 density on the McFarland scale) and one expander inoculated with a CoNS
suspension of 2.5 × 107 CFU/mL (CoNS; n = 10). Pressure/volume curves and histological and microbiological evaluations were performed. Operating room air
samples and contact skin samples were collected for microbiological evaluation. The rabbits were euthanized at four weeks.
Results: In the fibrin group, significantly decreased intracapsular pressures, thinner capsules, loose/dense (<25%) connective tissue, and negative/mild angiogenesis
were observed. In the CoNS group, increased capsular thicknesses and polymorph-type inflammatory cells were the most common findings. Similar bacteria in capsules,
implants, and skin were cultured from all the study groups. One Baker grade IV contracture was observed in an implant infected with Micrococcus spp.
Conclusions: Fibrin was associated with reduced capsule formation in this preclinical animal model, which makes fibrin an attractive potential
therapeutic agent in women undergoing breast augmentation procedures. Clinical strategies for preventing bacterial contamination during surgery are
crucial, as low pathogenic agents may promote CC.
Keywords
capsule, tissue expander, breast implants, histology, microbiology, coagulase-negative staphylococci, fibrin
Accepted for publication August 25, 2010.
The investigation of various possible mechanisms for capsular contracture (CC) has been complicated by the lack of
standardized animal models. Multiple reports have described
the effects of bacteria on CC, and one report exists on the
possible role of fibrin, but no single study has compared
these parameters at one experimental time point.
There is evidence that bacterial colonization of breast
implants is partially responsible for CC, and coagulasenegative staphylococci (CoNS; particularly Staphylococcus
epidermidis) have been largely implicated.1-15 Adams et
al16,17 showed that S. epidermidis colonization of breast
implants was more likely to result from bacterial contamination at the time of implantation than from ongoing
contamination from the adjacent ductal system. Because
of the low pathogenicity of CoNS and the existence of
microorganisms in a dormant phase within the biofilm
formed around the implant, CC does not usually clinically
Dr. Marques and Prof. Amarante are from the Department of
Surgery, Faculty of Medicine, University of Oporto and from the
Department of Plastic and Reconstructive Surgery, Hospital of
São João, Portugal. Dr. Brown is from the Department of Plastic
Surgery Research, Nancy L. & Perry Bass Advanced Wound Healing
Laboratory, UT Southwestern Medical School at Dallas, Texas, USA.
Prof. Cordeiro and Prof. Morales-Helguera are from the Department
of Chemistry, Faculty of Sciences, University of Oporto, Portugal.
Dr. Rodrigues-Pereira is from the Department of Pathology, Hospital
of São João, Oporto, Portugal. Prof. Gonçalves-Rodrigues and
Dr. Cobrado are from the Department of Microbiology, Faculty of
Medicine, University of Oporto, Portugal. Dr. Queirós, Dr. Luís, and
Dr. Freitas are from the Department of Experimental Surgery, Faculty
of Medicine, University of Oporto, Portugal.
Corresponding Author:
Dr. Marisa Marques, Hospital de São João, Serviço de Cirurgia Plástica,
Alameda Prof. Hernâni Monteiro, 4202 Porto, Portugal.
E-mail: [email protected]
Downloaded from aes.sagepub.com at ASAPS - Residents on May 19, 2011
Marques et al
421
manifest until some remote time after placement of breast
implants.18-22
Fibrin glue consists of two components, a fibrinogen solution and a thrombin solution rich in calcium. Fibrin serves as
a binding reservoir for several growth factors, such as vascular endothelial growth factor (VEGF),23 transforming growth
factor–β1,24 and basic fibroblastic growth factor (bFGF).25
Fibrin glue has been studied for decades for its applications
both in a surgical setting and as an hemostatic and sealant
agent. It is routinely used in gastrointestinal anastomosis,
breast surgery, facelifts, abdominoplasty, nerve repairs, graft
securing, neurosurgery, and ophthalmology.26-36 More
recently, it has also gained attention as a possible delivery
mechanism for drug therapies.37 For example, in a study by
Zhibo and Miaobo,38 release of lidocaine through fibrin glue
was tested for pain reduction in breast augmentation
patients. Patients who received fibrin glue with lidocaine
in the subpectoral pocket experienced less pain than those
who received the same amount of lidocaine or fibrin glue
alone. In another study,39 we applied an autologous fibrin
to the implant pocket as a contracture-inducing agent and
compared the results to a control group. The degree of
fibrosis was greater in the fibrin-exposed groups in both
the rabbit and human components of the study. Importantly,
there was a significant increase in intracapsular pressure
in the fibrin-exposed group. However, in another preclinical study40 with fibrin (Tisseel/Tissucol; Baxter US,
Deerfield, Illinois) sprayed onto the tissue expander and
capsule pocket, a significant decrease in intracapsular
pressures was found in the experimental fibrin group as
compared to a control group at four weeks. For both the
control and fibrin groups, mixed inflammation was correlated with decreased intracapsular pressure, whereas
mononuclear inflammation was correlated with increased
intracapsular pressure. The predominant isolate in capsules, tissue expanders, and rabbit skin was CoNS.
The purpose of this study was to perform a comprehensive evaluation, in a New Zealand white rabbit model, of the
relationships among intracapsular pressure (recorded directly
by a tissue expander), histological characteristics, and infection of breast implants. Microbiological analysis of rabbit skin
and operating room air was performed to account for contamination during the surgical procedure. Our aim was to
provide research data that could be translated into clinical
practice. Therefore, we elected to (1) apply a commercial
fibrin product (Tisseel/Tissucol) into the breast implant
pocket to clarify the effect on capsule formation and (2)
assess implants contaminated directly with CoNS, which
were previously reported as CC-inducing agents. At present,
there are no reports examining these two variables with a
preclinical animal protocol for direct comparison.
Methods
Thirty-one New Zealand white female rabbits (3-4 kg)
were each implanted with one 20-cc textured tissue
expander (Allergan, Inc., Santa Barbara, California) and
two textured breast implants (90 cc; Allergan, Inc., Santa
Barbara, California), according to approved institutional
animal care protocol. Prior to surgery, the skin of each
rabbit was washed with Betadine surgical scrub (Purdue
Pharma LP, Stamford, Connecticut), which contains 7.5%
povidone-iodine, and their skin was disinfected with
Betadine solution, which contains 10% povidone-iodine.
The surgical procedure was performed in an animal operating theater following aseptic rules. Penicillin G 40,000
U/kg intramuscularly (IM) was administered intraoperatively. Talc-free gloves were used at all times during the
procedure.
Implant pockets were developed in the subpanniculus
carnosis along the back region, with atraumatic dissection.
Under direct vision, particular attention was paid to
hemostasis, avoiding blunt instrumentation; there was no
obvious bleeding. A sterile dressing was placed over the
skin around the incision before the tissue expander and
the implants were inserted to eliminate contact with the
skin.41 A new pair of talc-free gloves was worn when
inserting the tissue expander and the implants.
The rabbits were divided into three groups: (1) those
that received untreated implants and expanders (control;
n = 10), (2) those that received two implants sprayed with
2 mL of fibrin and one expander sprayed with 0.5 mL of
fibrin (fibrin; n = 11), and (3) those that received two
implants inoculated with 100 µL of a CoNS suspension
(108 CFU/mL—0.5 density on the McFarland scale) and
one expander inoculated with a CoNS suspension of 2.5 ×
107 CFU/mL (CoNS; n = 10).
All rabbits were sacrificed at four weeks. Prior to sacrifice, each animal was anesthetized, and the dorsal back
area was shaved. A pressure-measuring device (Stryker
Instruments, Kalamazoo, Michigan) was connected to the
tissue expander port, and intracapsular pressures were
recorded in 5-mL increments prior to any incision in the
capsule. All capsule samples were submitted for histological and microbiological evaluation. All implants and
expander devices were also submitted for microbiological
evaluation.
Microbiological Assessments
Air. Operating room air samples (n = 36) were collected as described in our previous study40 with a MAS
100-Eco air sampler (EMD Chemicals, Inc., Gibbstown,
New Jersey) at a flow rate of 100 L per minute. Identification of bacterial and fungal isolates followed standard
microbiological procedures. Gram-positive cocci were
characterized by biochemical methods. Catalase-positive
and coagulase-positive colonies were identified as Staphylococcus aureus; catalase-positive and coagulase-negative
colonies were identified as coagulase-negative staphylococci. Gram-negative bacilli were characterized with Vitek
2 software (VT2-R04.02, bioMérieux, Inc., Durham, North
Carolina). Fungi were characterized following their macroscopic appearance and microscopic morphology.
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422
Aesthetic Surgery Journal 31(4)
Rabbit skin. A total of 93 contact plates (31 brain-heart
agar, 31 mannitol salt agar, and 31 Sabouraud agar) were
pressed to the shaved dorsal skin surfaces, also as described
in our previous study.40 Brain-heart and mannitol salt agar
plates were incubated for three days at 28°C; Sabouraud
plates were incubated for seven days at 28°C. Bacterial and
fungal colonies were counted and reported as cfu/cm2. The
identification of the bacteria and fungi followed the procedures reported above.
Capsules/implants/tissue expanders. Excised implants, tissue
expanders, and representative capsule samples were incubated at 37°C for three days in brain-heart broth and examined
daily; changes in turbidity of the broth media were considered positive and were subcultured in solid agar media.
Characterization of microbial isolates followed the procedures
described in the section on skin testing.
Histological Assessment
Capsule specimens were fixed with 10% buffered formalin
and embedded in paraffin. Sections were stained with
hematoxylin and eosin and histologically evaluated for tissue inflammation and capsular thickness. The type and
intensity of the inflammatory infiltrate were analyzed.
Inflammation was grouped into three categories: mononuclear/chronic (lymphocytes, plasmocytes, and histiocytes),
mixed/subacute (mononuclear cells and eosinophils), and
polymorph/acute (eosinophils and heterophils). Inflammatory
infiltrate intensity was categorized according to the following criteria: absent (−), mild (+), moderate (++), and
severe (+++).40,42
Samples were stained with Masson’s trichrome to characterize the connective tissue (loose or dense), organization of the collagen fibers (arranged in a parallel array or
haphazard), angiogenesis (absent, mild, moderate, or
high), and fusiform cell density (mild, moderate, or high).
The density of the connective tissue was semiquantitatively separated into one of four groups: (1) less than 25%,
(2) 25% to 50%, (3) 50% to 75%, and (4) >75%.
Histological sections were reviewed and graded by a
pathologist blinded to the protocol.
Statistical Analysis
Data were grouped according to the type of product
applied to the breast implants: control (none; n = 10 rabbits, 20 implants), CoNS (n = 10 rabbits, 20 implants), or
fibrin (n = 11 rabbits, 22 implants). One-way analysis of
variance test—either parametric or nonparametric
(Kruskal-Wallis H test)—was performed to determine
whether the continuous variables (intracapsular pressure
and histologically measured thickness) were equal, followed by post hoc range tests to identify homogeneous
subsets across groups. Two-tailed independent paired t
tests were used, along with the nonparametric alternative
Mann-Whitney U tests. Categorical variables were evalu-
Figure 1. Pressure-volume curves. Note the significant
difference in intracapsular pressure in the fibrin group.
CoNS, coagulase-negative staphylococci.
ated by chi-square statistics and by phi, Cramer’s V, and
contingency coefficients. Statistical significance was calculated at p ≤ .05. Major trends within each group were
further examined with the Chi-squared Automatic
Interaction Detection (CHAID) method,43 using the likelihood ratio chi-square statistic as growing criteria, along
with a Bonferroni 0.05 adjustment of probabilities. All
analyses were carried out with the Statistical Package for
Social Sciences Version 16 (SPSS, Inc., an IBM Company,
Chicago, Illinois).
Results
Statistical analyses revealed no significant differences in
histological and microbiological results between breast
implants and tissue expanders. Because no differences
were found, these data are not shown.
Intracapsular Pressure
During pressure measurements, five (50%) capsules ruptured in the control group, and five (50%) capsules ruptured in the CoNS group. To avoid a prohibitively small
sample size, the ruptured capsules were not excluded from
our statistical analyses, but the pressure value measured
before rupture was maintained after further additional milliliters of saline were added. Significantly decreased intra­
capsular pressures were registered for the fibrin group as
compared to the control and the CoNS groups (p ≤ .001
and p ≤ .05, respectively; Figure 1). Statistical analyses
revealed no significant differences between the CoNS and
the control groups.
Histology
Average capsular thicknesses were 0.81 ± 0.21 mm, 0.47 ±
0.13 mm, and 1.06 ± 0.29 mm in the control, fibrin, and
CoNS groups, respectively. Capsular thickness was not
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Marques et al
A
423
B
PRESSURE
≤ 173: 12 (60%)
>173: 8 (40%)
PRESSURE
° 140: 14 (63.6%)
<140: 8 (36.4%)
THICKNESS p < 0.004
THICKNESS p < 0.002
≤ 0.8 mm
≤ 173: 12 (80%)
> 173: 3 (20%)
> 0.8 mm
≤ 173: 0 (0%)
> 173: 5 (100%)
≤0.5 mm
° 140: 12 (85.7%)
<140: 2 (14.3%)
>0.5 mm
° 140: 2 (25%)
<140: 6 (75%)
Figure 2. Decision tree by Chi-squared Automatic Interaction Detection (CHAID) algorithm for pressure and thickness. (a)
Control group and (b) fibrin group.
statistically homogeneous across the three groups (p ≤
.001). Three subsets of similar means were found after
applying post hoc range tests: the first one comprised the
fibrin group (with the thinnest capsule), the second comprised the control group, and the third comprised the
CoNS group (with the thickest capsule). CHAID statistical
modeling showed a correlation between intracapsular
pressure measured at 20 mL and thickness for the control
and the fibrin groups (Figure 2). Decreased intracapsular
pressure was associated with thinner capsules for both
groups, and the converse was also true.
A mixed type of inflammatory cells was the most common finding in the control and fibrin groups, but in the
CoNS group, the polymorph type of inflammatory cells
was predominant (Table 1). For types of cells, significant
differences were observed between the control and CoNS
groups (p = .0001), as well as between the CoNS and
fibrin groups (p = .0009), but not between the control and
the fibrin groups. Intensity of inflammation was moderate
in the control and fibrin groups and mild in the CoNS
group (Table 1). Significant differences were also found
with regard to inflammatory intensity between the control
and CoNS groups (p = .011), as well as between the CoNS
and the fibrin groups (p = .0058), but not between the
control and the fibrin groups. Significant correlations
between the intensity of inflammation and the type of
inflammatory cells were observed for the control (p =
.005) and the fibrin (p = .006) groups.
Fibrosis was detected in all capsules; no significant differences regarding the fusiform cell density were observed
in any of the groups. With regard to connective tissue,
significant differences were found between the control and
fibrin groups (p = .005) and between the CoNS and fibrin
groups (p = .0007), with dense (>25%) connective tissue
in the control and the CoNS groups and ≤25% connective
tissue in the fibrin group.
Significant differences in the organization of the collagen fibers were observed between the control and fibrin
groups (p = .019) and between the CoNS and fibrin
groups (p = .0039), with haphazard collagen fibers in the
control and CoNS groups and fibers arrayed in parallel in
the fibrin group. Significant differences in angiogenesis
were found between the control and fibrin groups (p = .003)
and between the CoNS and the fibrin groups (p = .016),
Table 1. Outcomes for Capsule Inflammation in Control vs Experimental
Groups
Group
Type of
Inflammatory Cells
Control
Mononuclear
%
Polymorph
Fibrin
CoNS
Intensity
25.0
0
%
Mild
30.0
Moderate
70.0
Mixed
75.0
High
0
Mononuclear
13.6
Mild
31.8
Polymorph
13.6
Moderate
59.1
Mixed
72.8
High
9.1
Mononuclear
35.0
Mild
70.0
Polymorph
50.0
Moderate
30.0
Mixed
15.0
High
0
CoNS, coagulase-negative Staphylococci.
with moderate/high in the control and CoNS groups and
negative/mild in the fibrin group.
Microbiology
Bacteria were isolated in 31% (19 of 62) of the removed
capsules and in 84% (56 of 62) of the removed implants
(Table 2). The predominant isolates were CoNS, which
were found in 16% of all culture-positive capsules (10
of 62) and in 60% of culture-positive implants (37 of
62). Overall, 97% of culture-positive capsules and 90%
of culture-positive implants yielded a single isolate,
whereas 3% and 10% (respectively) yielded two. No
bacteria were detected on 69% of the removed capsules
and on 16% of the removed implants. No fungi were
recovered from the removed capsules or implants among
all groups.
Statistical analysis revealed no significant differences
in the type of bacteria or in the frequency of culture
positivity among the study groups. Also, there was no
significant association between microbial presence and
histological data.
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424
Aesthetic Surgery Journal 31(4)
Table 2. Bacteria Isolated From Capsule and Implant Samples Removed
From All Sacrificed Rabbitsa
Number (%) of Positive Cultures
b
Capsules
Implants
Bacteria
Group
Coagulasenegative
staphylococci
(CoNS)
Control
2 (10)
13 (65)
Fibrin
2 (9)
9 (41)
CoNS
6 (30)
15 (75)
Control
2 (10)
2 (10)
Fibrin
1 (5)
7 (32)
CoNS
0 (0)
2 (10)
Control
1 (15)
1 (5)
Fibrin
3 (14)
4 (18)
CoNS
0 (0)
2 (10)
Control
0 (0)
0 (0)
Fibrin
0 (0)
0 (0)
CoNS
2 (10)
1 (5)
Staphylococcus
aureus
Bacillus grampositive
Micrococcus spp.
a
Sixty-two capsule samples and 62 implant samples were obtained from 31 rabbits.
Control (10 rabbits; 20 capsules and 20 implants), fibrin (11 rabbits; 22 capsules and 22
implants), and CoNS (10 rabbits; 20 capsules and 20 implants).
b
Similar bacteria were isolated from the rabbit skin. The
predominant isolates were CoNS, which was found in 37 of
all 45 sacrificed rabbits (82%), followed by gram-positive
bacilli (60%), S. aureus (33%), and Micrococcus spp. (9%).
Other isolates found were Enterococcus hermannii and
Proteus mirabilis, although all occurred much less frequently
than the others listed previously. No skin sample was culture
negative; 35 samples yielded more than one isolate. The
bacterial isolates from rabbit skin were similar to those from
the removed capsules and implants. Finally, CoNS were also
cultured from all of the air samples; other airborne isolates
were gram-positive and gram-negative bacilli such as Micrococcus spp., Cryptococcus laurentii, Acinetobacter lwoffii, and
Enterococcus agglomerans. Fungal species such as Penicillium
spp., Aspergillus niger, Aspergillus flavus, and Aspergillus
fumigatus were recovered from the operating room air, with
Penicillium being the most common fungal isolate.
In the CoNS group, one animal developed a Baker
grade IV contracture44 in one breast implant (Figure 3a).
The capsular thickness measured 1.70 mm and was the
largest among all capsules (Figure 3b). The type of inflammatory cells was polymorphous, with moderate intensity.
Histological evaluation of fibrosis revealed 25% to 50%
connective tissue density, haphazard collagen fibers, and
moderate angiogenesis. The capsule and breast implant
were both infected with a Micrococcus spp. isolate; no
other bacteria or fungi were detected.
Discussion
Significant results were demonstrated in each of our experimental groups. In the fibrin group, the data showed significantly decreased intracapsular pressures and capsular
thicknesses without any capsule rupture, as compared to
the control and CoNS groups. For the fibrin and control
groups, decreased intracapsular pressures were correlated
with thinner capsules. In terms of inflammation, mixedtype inflammatory cells were the most common finding for
both fibrin and control groups. In the fibrin group, ≤25%
connective tissue density was observed, as compared to the
control and the CoNS groups, which had >25% connective
tissue density. In the fibrin group, negative/mild angiogenesis was observed; the control and the CoNS groups had
moderate/high angiogenesis. No significant differences
Figure 3. (a) The one case of capsular contracture (Baker grade IV) is shown. (b) An extremely thick and opaque capsule was
evident in the implant associated with the contracture.
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Marques et al
425
regarding fusiform cell density were observed between the
fibrin and control groups.
In the CoNS experimental group, capsular thickness
was increased as compared to the control. Polymorph-type
inflammatory cells were the most common observation in
the CoNS group, which was significantly different from
the control group. Regarding fusiform cell density, connective tissue characteristics, organization of the collagen
fibers, and angiogenesis, similar results were observed for
both CoNS and control groups.
Similar bacterial isolates were observed among all the
study groups for both implants and capsules. Implants were
2.7 times more frequently infected than capsules. The predominant isolates were CoNS, which were present 3.8 times
more frequently in implants than in capsules. There was no
significant association between microbial presence and histological data. Bacteria isolates from rabbit skin were similar to
those isolated from capsules and implants. As expected, the
predominant isolate in rabbit skin, as in implants and capsules, was CoNS. Unexpectedly, Micrococcus spp. was isolated from rabbit skin specimens, operating room air samples,
and one rabbit; in that specific rabbit, Micrococcus spp. was
detected on the capsule but not on the implant surface, and
this capsule did not develop CC. Interestingly, on the contralateral implant in the same rabbit, a Micrococcus spp. isolate
was detected on both the implant surface and the capsule,
which demonstrated a Baker grade IV contracture.44 To the
best of our knowledge, this is the first report that shows a
direct association between the presence of Micrococcus spp.
and clinical CC in a rabbit model. Fungi were isolated from
the operating room air samples but not from the rabbit skin,
capsules, or implants. As far as we know, this is also the first
report examining microbial cross-contamination among air,
rabbit skin, capsules, and implants.
Our results support the probable role of fibrin as an
agent that may modify capsule formation and mitigate
subsequent CC since it is associated with decreased capsule thickness and pressure, ≤25% connective tissue density, and negative/mild angiogenesis. The decreased
intracapsular pressure and thinner capsules were also consistent with other clinical reports of CC.42,45-47 The relationship of CC to dense collagen and increased angiogenesis
has already been demonstrated in other reports45,48,49; this
was also found in our control and CoNS groups. The relationship between the organization of the collagen fibers
(parallel or haphazard) and CC is controversial; our results
are similar to a study from Karaçal et al.47
The cytokine-transforming growth factor beta 1 (TGFβ1) is a central mediator of fibrosis.50-52 Some reports have
focused on fibrin’s properties for enhanced wound healing
through the reduction of collagen extracellular matrix and
decreased TGF-β1.53-56 The TGF-β1-inhibitor peptide was
shown to be significantly effective in achieving a reduction
in fibrosis in silicone breast implants.57 The use of fibrincontaining preparations (Tisseel and Vi-Guard, Melville
Biologics, Inc, Melville, New York) allows for the closure of
dead space and approximation of the skin flaps, and it has
been argued that fibrin-containing tissue adhesives produce
a dense architecture that inhibits angiogenesis and vascular ingrowth.58 To the best of our knowledge, this is the
first preclinical study with a commercial fibrin compound
(Tissucol/Tisseel) applied to a textured silicone breast
implant.
According to our results, bacterial infection of breast
implants was more common than capsule infection, and the
predominant isolates were CoNS. This is consistent with the
fact that CoNS, a commensal bacteria of the skin, is the predominant cause of biomaterial-associated infection, commonly mediated by formation of biofilms.18-21,59,60 The major
pathogenicity is related to extensive biofilm formation on
solid surfaces, which is extremely difficult to treat with antibiotics, thereby necessitating invasive procedures to remove
the infected tissue or devices.61-63 A strong correlation
between the presence of biofilm (particularly by S. epidermidis) and significant CC was reported by Pajkos et al.22
They assumed that biofilm on the outer surface of the
implant, once established, acts as a focus of irritation and
chronic inflammation, leading to accelerated CC.22 However,
our results are contradictory to that report.22 In the Pajkos et
al22 study, the rate of recovery from bacteria from the implant
surface was lower than the rate of recovery from the capsule
surface, but the authors explain that there was a greater sensitivity in detecting bacterial growth on capsules.
The Baker grade IV contracture in our study, which
occurred in the implant with the thickest capsule, was
unusual in that contracture developed quickly with an
acute inflammation. Unexpectedly, both the capsule and
implant were infected only with Micrococcus spp., a low
pathogenic agent. As far as we know, there are few reports
concluding that Micrococcus spp. may have a true etiologic
role in infection64 or that it is mediated by formation of
bacterial biofilms.65,66
Our fibrin results are contradictory to one of our previous reports39 but consistent with our previous preclinical
study.40 This may be explained by the product we applied.
In the first study,39 we applied an autologous fibrin glue of
unknown fibrin concentration into the implant pocket; in
the current experimental design and the previous preclinical study,40 we sprayed a commercial fibrin product widely
studied and used in clinical practice in Europe and the
United States to reduce polypropylene meshes adhesions,67,68 the incidence of posterior spinal epidural adhesion formation,30 and the recurrence rate of pterygium
after surgery.36 Another explanation may lie in the application mechanism (manual with a syringe in the first study
vs sprayed in the latter two). A previous study found that
a thin layer of glue is preferable to a thick one69; a thin
layer of fibrin glue (such as would occur with a spray)
may support the healing process, whereas a thick layer of
adhesive inhibits skin graft healing.70 Also, in this study,
capsule pressure was measured directly in the tissue
expanders to achieve more accurate results.47
Fibrin glue has been shown to act as an hemostatic
agent,71 an agent for enhanced wound healing by the
reduction of collagen extracellular matrix and decreased
TGF-β1 (a mediator of fibrosis),53-56 an agent for adhesion
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426
Aesthetic Surgery Journal 31(4)
prevention,67,68 a widely used ophthalmology tool,31-36 and
a drug delivery system for antibiotics.72 Fibrin glue was
also specifically tested in a clinical model as a drug delivery system following breast augmentation.38 Our preclinical animal model results also show it to be a promising
agent for the prevention of CC.
The limitation of this study was the insertion of only one
tissue expander per rabbit, which allowed for direct intercapsular pressure measurement through the port. To correlate
intracapsular pressure from tissue expanders with histological and microbiological results from breast implants, we
performed statistical analyses that revealed no significant differences in histological and microbiological results between
breast implants and tissue expanders. It would have been
better to measure the pressure directly through 90-cc silicone
breast implants with ports to achieve more accurate results,
but these are not commercially available. One strength of this
study was the statistical analyses of the data among the three
groups using the CHAID method, a sophisticated algorithm
used in many other disciplines that allows the investigator to
adjust for the probability of a single variable among multiple
variables.
Future studies include a prospective clinical study comparing a female control group with an experimental group
that had Tissucol/Tisseel sprayed on the implant or
pocket, with a follow-up period longer than 42 months.73
A preclinical study analyzing S. epidermidis and
Micrococcus spp. biofilm development in silicone breast
implants where the ports have been sprayed with Tissucol/
Tisseel and infected with bacteria would also be helpful.
Conclusions
The results from this preclinical rabbit model suggest
that fibrin applied to the breast implant pocket may
reduce capsular contracture. Since their relatively safe
profile has been well established, fibrin-containing
compounds are therefore an attractive adjunct for use in
women undergoing breast augmentation. Clinical strategies for preventing bacterial contamination during surgery are crucial, as low pathogenic agents may promote
capsular contracture.
Acknowledgments
The authors thank Tom Powell, Fernando Carvalho, Pedro Lopes,
Luis Sogalho, Anabela Silvestre, Jiying Huang, Debby Noble,
James Richardson, Donna Henderson, Maria José Neto, Luis
Bastos, Pedro Leitão, Nuno Rego, Isabel Santos, Cristina Moura,
and Elisabete Ricardo for excellent assistance with organizing
much of this work, cleaning the operating room, taking air samples, and helping care for the rabbits. All were involved with the
surgeries. Dr. Carvalho was the veterinarian.
Disclosures
The authors declared no potential conflicts of interest with respect
to the research, authorship, and publication of this article.
Funding
Research support was provided by the Faculty of
Medicine-UP, the Faculty of Sciences-UP, the Hospital of São
João, Fundação Ilídeo Pinho, and Comissão de Fumento de
Investigação em Cuidados de Saúde Daniel Serrão at Portugal,
as well as the Department of Plastic Surgery Research–University of Texas Southwestern Medical Center, Dallas, Texas. Tissue
expanders and implant devices were supplied by Allergan, Inc.
(Santa Barbara, California), and Tissucol/Tisseel supplies were
provided by Baxter Healthcare (Deerfield, Illinois).
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Publication
Aesthetic Surgery Journal
http://aes.sagepub.com/
Animal Model of Implant Capsular Contracture : Effects of Chitosan
Marisa Marques, Spencer A. Brown, Pedro Rodrigues-Pereira, M. Natália, D. S. Cordeiro, Aliuska Morales-Helguera, Luís
Cobrado, Lara Queirós, Rui Freitas, João Fernandes, Inês Correia-Sá, Acácio Gonçalves Rodrigues and José Amarante
Aesthetic Surgery Journal 2011 31: 540
DOI: 10.1177/1090820X11411475
The online version of this article can be found at:
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ERN
INT ATI
IBUTION
TR
AL CON
ON
Research
Animal Model of Implant Capsular
Contracture: Effects of Chitosan
Aesthetic Surgery Journal
31(5) 540­–550
© 2011 The American Society for
Aesthetic Plastic Surgery, Inc.
Reprints and permission:
http://www​.sagepub.com/
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DOI: 10.1177/1090820X11411475
www.aestheticsurgeryjournal.com
Marisa Marques, MD; Spencer A. Brown, PhD;
Pedro Rodrigues-Pereira, MD; M. Natália, D. S. Cordeiro, PhD;
Aliuska Morales-Helguera, PhD; Luís Cobrado, MD; Lara Queirós, MD;
Rui Freitas, MD; João Fernandes, PhD; Inês Correia-Sá, MD;
Acácio Gonçalves Rodrigues, MD, PhD; and José Amarante, MD, PhD
Abstract
Background: The mechanism(s) responsible for breast capsular contracture (CC) remain unknown, but inflammatory pathways play a role. Various
molecules have been attached to implant shells in the hope of modifying or preventing CC. The intrinsic antibacterial and antifungal activities of chitosan
and related oligochitosan molecules lend themselves well to the study of the infectious hypothesis; chitosan’s ability to bind to growth factors, its
hemostatic action, and its ability to activate macrophages, cause cytokine stimulation, and increase the production of transforming growth factor (TGF)–β1
allow study of the hypertrophic scar hypothesis.
Objective: The authors perform a comprehensive evaluation, in a rabbit model, of the relationship between CC and histological, microbiological, and
immunological characteristics in the presence of a chitooligosaccharide (COS) mixture and a low molecular weight chitosan (LMWC).
Methods: Eleven adult New Zealand rabbits were each implanted with three silicone implants: a control implant, one impregnated with COS, and one
impregnated with LMWC. At four-week sacrifice, microdialysates were obtained in the capsule-implant interfaces for tumor necrosis factor alpha (TNF-α)
and interleukin-8 (IL-8) level assessment. Histological and microbiological analyses were performed.
Results: Baker grade III/IV contractures were observed in the LMWC group, with thick capsules, dense connective tissue, and decreased IL-8 levels
(p < .05) compared to control and COS groups. Capsule tissue bacterial types and microdialysate TNF-α levels were similar among all groups.
Conclusions: Chitosan-associated silicone implantation in a rabbit model resulted in Baker grade III/IV CC. This preclinical study may provide a model
to test various mechanistic hypotheses of breast capsule formation and subsequent CC.
Keywords
breast implants, capsular contracture, chitosan, microdialysis, histology, microbiology, immunology
Accepted for publication August 10, 2010.
Dr. Marques, Dr. Correia-Sá and Prof. Amarante are from the Department of Surgery, Faculty of Medicine, University of Oporto and from the Department
of Plastic and Reconstructive Surgery, Hospital of São João, Portugal. Dr. Brown is from the Department of Plastic Surgery Research, Nancy L. & Perry
Bass Advanced Wound Healing Laboratory, UT Southwestern Medical School at Dallas, Texas, USA. Prof. Cordeiro and Prof. Morales-Helguera are from
the Department of Chemistry, Faculty of Sciences, University of Oporto, Portugal. Dr. Rodrigues-Pereira is from the Department of Pathology, Hospital
of São João, Oporto, Portugal. Prof. Gonçalves-Rodrigues and Dr. Cobrado are from the Department of Microbiology, Faculty of Medicine, University of
Oporto, Portugal. Dr. Queirós and Dr. Freitas are from the Department of Experimental Surgery, Faculty of Medicine, University of Oporto, Portugal, and
Prof. Fernandes is from the Biotechnology School, University of Oporto, Portugal.
Corresponding Author:
Dr. Marisa Marques, Faculty of Medicine, University of Porto, Hospital de São João, Serviço de Cirurgia Plástica (piso 7), Alameda Prof.
Hernâni Monteiro, 4202-451 Porto, Portugal.
E-mail: [email protected]
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Marques et al
541
The true etiology of breast capsular contracture (CC) associated with implant devices, along with the most appropriate course of treatment, remains elusive despite extensive
study. Two prevailing theories have emerged in the literature1-18: the infectious hypothesis and the hypertrophic
scar hypothesis. As a solution, various molecules have
been applied to implant shells in the hope of modifying or
preventing CC. Chitin, the polymer D-glucosamine in β
(1,4) linkage, is the major component of the exoskeletons
of crustaceous and cell wall fungi.19 Chitosan is a
deacetylated product of chitin. In the literature, the term
chitosan is used to describe chitosan polymers with different molecular weights (50-2000 kDa), viscosities, and
degrees of deacetylation (40%-98%).20 Material with lower
levels of deacetylation degrades more rapidly.21-23 Chitosan
has been a better-researched version of the biopolymer
because of its ready solubility in dilute acids, which makes
it more accessible for utilization and chemical reactions.24
Chitooligosaccharides (COS) are degraded products of chitosan, or the deacetylated and degraded products of chitin,
by chemical and enzymatic hydrolysis.
Chitosan and related oligochitosan molecules have
intrinsic antibacterial and antifungal activities25-28 that
lend themselves well to the study of the infectious hypothesis. Furthermore, chitosan’s ability to bind to growth factors,29,30 its hemostatic action,31 and its ability to activate
macrophages, cause cytokine stimulation,31 and increase
the production of transforming growth factor (TGF)–β132
permit study of the hypertrophic scar hypothesis.
Data from a study by Khor and Lim,24 which included
cell cultures and an animal model, indicated that chitin and
chitosan processed in different shapes and in combination
with different materials were noncytotoxic. The authors suggested that inclusion of these materials might yield tissueengineered implants that would be biocompatible and viable.
These attributes make chitosan a promising biopolymer for
modulating wound healing (full-thickness skin defects and
dermal burns)26,29,33 and for use in orthopedics (cartilage,
anterior cruciate ligament, intervertebral disk, bone, osteomyelitis)28,34 and otologic diseases (tympanoplasty).35
Fibrosis is a major global health problem, but its etiology,
pathogenesis, diagnosis, and therapy have yet to be addressed.
Fibrosis can occur as a consequence of many pathologic
conditions: (1) spontaneously (keloids, Dupuytren’s contracture), (2) from tissue damage (postoperative adhesions,
burns, alcoholic and postinfection liver fibrosis, silica dust,
asbestos, antibiotic bleomycin), (3) as a result of inflammatory disease (infections, scleroderma), (4) in response to
foreign implants (breast implant capsular contracture, cardiac
pacemakers), and (5) from tumors (fibromas, neurofibromatosis). The early stages of fibrotic conditions are characterized by a perivascular infiltration of mononuclear cells and
the subsequent imbalance of anti- and profibrotic cytokine
profiles. One of the most prominent activators of mononuclear cells and fibroblasts is hyaluron fragments, which not
only induce the expression of various cytokines (interleukin
[IL]–1, IL-12, and tumor necrosis factor alpha [TNF-α]),
chemokines (MPI-1A, MCP-1, IL-8), and inducible nitric
oxide synthase (iNOS) but also trigger the expression and
secretion of macrophage-derived matrix metalloproteinases
(MMP), enzymes essential for extracellular matrix (ECM)
cleavage.36 IL-8 is a neutrophil chemoattractant factor. Levels
of IL-8 are increased in scleroderma skin biopsy specimens.37
Cultured scleroderma dermal fibroblasts make more IL-8
than normal fibroblasts. Studies in animal models of pulmonary fibrosis have shown the importance of chemokines in
promoting angiogenesis, which is necessary for the development of pulmonary fibrosis.
Clues about the potential role of IL-8 in fibrosis come
from studies of patients with idiopathic pulmonary fibrosis.38 A low concentration of TNF-α increases fibroblast
proliferation, whereas high TNF-α concentration decreases
fibroblast proliferation.39 However, TNF-α levels are markedly elevated in liver fibrosis, considered a profibrinogenic
cytokine such as TGF-β1.40 The immune inflammatory
response and macrophage release of IL-8 and TNF-α
induced by phagocytosis of periprosthetic wear debris
stimulate bone reabsorption at implant or cement-bone
interface. These cytokines directly induce fibroblast proliferation and tissue necrosis. Increased concentrations of
IL-8 and TNF-α in the peripheral circulation of patients
with large joint prostheses would indicate aseptic loosening.41 As far as we know, there are no reports correlating
capsule formation or CC with TNF-α and IL-8 levels. For
all of these reasons, we believe that studying the role of
these markers in capsule formation is important to the
literature.
Microdialysis enables measurement of the molecules in
the extracellular fluid around the capsule. Originally initiated more than 30 years ago,42 microdialysis studies in
humans have been mainly limited to head injury,43-46 subarachnoid hemorrhage,47 epilepsy,48 and cerebral tumors.49,50
IL-8 and TNF-α are known major biomarkers for inflammation,51-53 which can be examined through microdialysis. To
date, no preclinical model has been reported to assess possible environmental challenges that may prevent or modulate the wound-healing response with chitosan and related
oligochitosan molecules associated with silicone implants.
Therefore, we performed a comprehensive evaluation, in a
rabbit model, of the relationships among CC rates and histological, microbiological, and immunological characteristics in the presence of COS mixtures and low molecular
weight chitosan (LMWC). To monitor levels of inflammatory biomarkers in the breast capsule extracellular fluid,
IL-8 and TNF-α were determined with the microdialysis
technique.
Methods
Eleven New Zealand white female rabbits (3-4 kg) were
each implanted with three different textured breast
implants, according to an approved institutional animal
care protocol. The implants were each 90 cc and were
provided by Allergan, Inc. (Santa Barbara, CA). Prior to
surgery, the skin of each rabbit was washed with Betadine
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542
Aesthetic Surgery Journal 31(5)
Figure 1. (A) Rabbit is shown with control implant, chitooligosaccharide (COS) implant, and chitosan implant (contracture
grade IV). (B) The chitosan implant is pictured. Baker Grade IV contracture is evident. (C) The chitosan implant’s extremely
thick, dense, and opaque capsule can be seen. (D) Hematoxylin and eosin stain of the chitosan implant, at ×100 magnification,
with apoptotic cells. These cells have hyperchromatic and fragmented nuclei.
surgical scrub (Purdue Pharma LP, Stamford, Connecticut)
that contained 7.5% povidone-iodine, and their skin was
disinfected with Betadine solution that contained 10%
povidone-iodine. The surgical procedure was performed in
an animal operating theater following aseptic rules.
Penicillin G 40,000 U/kg intramuscularly (IM) was administered intraoperatively. Talc-free gloves were used at all
times during the procedure.
Implant pockets were developed in the subpanniculus
carnosis along the back region, with atraumatic dissection.
Under direct vision, particular attention was paid to
hemostasis, avoiding blunt instrumentation; there was no
obvious bleeding. A sterile dressing was placed over the
skin around the incision before the tissue expander and
the implants were inserted to eliminate contact with the
skin.54 A new pair of talc-free gloves was worn when
inserting the tissue expander and implants.
Each implant was placed beneath the panniculus carnosis along the back (Figure 1A). Each rabbit received an
untreated implant (control), an implant impregnated with
COS (molecular weight [MW] 1.4 kDa; Nicechem,
Shanghai, China), and an implant impregnated with
LMWC (MW 107 kDa; Sigma-Aldrich, Sintra, Portugal).
Both chitosan mixtures possessed a deacetylation degree
in the range of 80% to 85%. Implants were prepared by
immersion in either COS (20.0 mg/mL) or LMWC (10.0
mg/mL) solutions with pH adjusted to 5.8 to 5.9 for two
hours. Implants were incubated at 37 °C in a flow chamber for two days, then packed and sterilized by ethylene
oxide.
Rabbits were sacrificed at four weeks. Prior to sacrifice,
each animal was anesthetized, and a 5-mm incision was
made directly over the implant, through the skin, panniculus
carnosus, and capsule. A 100,000-MW cutoff microdialysis
probe (CMA Microdialysis, Stockholm, Sweden) was placed
by the capsule-implant interface, and microdialysates were
collected with sterile, normal saline solution (6 µL/min)
for one hour. Whole blood was obtained by venipuncture,
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Marques et al
543
and serum was collected after centrifugation (2000 g
min−1, 4 °C). Capsule samples were submitted to histological and microbiological evaluations.
Histological sections were reviewed and graded by a
pathologist blinded to the protocol.
Microdialysis Assessment
Microbiological Assessments
Air. Operating room air samples (n = 20) were collected
during all surgical procedures with the MAS 100-Eco air
sampler (EMD Chemicals, Inc., Gibbstown, New Jersey) at a
flow rate of 100 L/min. Identification of bacterial and fungal
isolates followed standard microbiological procedures. Grampositive cocci were characterized by biochemical methods.
Catalase-positive and coagulase-positive isolates were reported
as Staphylococcus aureus; catalase-positive and coagulasenegative isolates were reported as coagulase-negative
staphylococci. Gram-negative bacilli were characterized with
Vitek 2 software (VT2-R04.02; bioMérieux, Inc., Durham,
North Carolina). Fungi (molds) were characterized according
to their macroscopic and microscopic morphology.
Rabbit skin. A total of 33 contact plates (11 brain-heart
agar, 11 mannitol salt agar, and 11 Sabouraud agar contact
plates) were pressed to the shaved dorsal skin surfaces.
Brain-heart and mannitol salt agar plates were incubated
for three days at 28°C; Sabouraud plates were incubated
for seven days at 28°C. Bacterial and fungal colonies were
counted and reported as cfu/cm2. The identification of the
bacteria and fungi followed the procedures reported above.
Capsules and implants. Excised implants and representative capsule samples were incubated at 37°C for three days
in brain-heart broth and examined daily; changes in turbidity of the broth media were considered positive and were
subcultured in solid agar media. Characterization of microbial isolates followed the procedures described above.
Histological Assessment
Capsule specimens were fixed with 10% buffered formalin
and embedded in paraffin. Sections were stained with
hematoxylin and eosin and evaluated histologically for tissue inflammation and capsular thickness. Inflammatory
cells were grouped into three categories by type: (1)
mononuclear (lymphocytes, plasmocytes, and histiocytes),
(2) mixed (mononuclear cells and eosinophils), or (3)
polymorph (eosinophils and heterophils/neutrophils).
Inflammatory infiltrate intensity was categorized according to the following criteria: absent (−), mild (+), moderate (++), or severe (+++).55
Samples were stained with Masson’s trichrome56,57 to
characterize the organization of the collagen fibers
(arranged in a parallel array or haphazard), angiogenesis
(absent, mild, moderate, or high), and fusiform cell density (mild, moderate, or high). The dense connective tissue was semiquantitatively analyzed as (a) less than 25%,
with thick collagen bundles less than 25%; (b) 25% to
50%; (c) 51% to 75%; or (d) more than 75%.
TNF-α levels were determined with the manufacturer’s
instructions from a commercial kit (Invitrogen, Hu TNF-α
cat. no. KHC3014:1; Life Technologies, Inc., Carlsbad,
California). The assay was a solid-phase sandwich enzymelinked immunosorbent assay (ELISA) in which 100 µL of
microdialysis fluid was pipetted into each well. The protocol for IL-8 was performed with the BioSource Hu IL-8 US
kit (cat. no. KHC0083/KHC0084; Life Technologies, Inc.).
Statistical Analysis
Data were grouped according to the type of product
applied to the implant: control (none), COS, and LMWC
(chitosan). Group data were also analyzed separately for
the 11 sacrificed rabbits at four weeks after surgery. A twotailed paired t-test and the nonparametric alternative
Wilcoxon signed rank tests were applied to determine
whether continuous variables (histologically measured
thickness and dialysate levels of IL-8 and TNF-α) were
significantly different among control and experimental
groups. Categorical variables were evaluated by chi-square
statistics and by phi, Cramer’s V, and contingency coefficient tests. Statistical significance was presumed at p ≤ .05.
Major trends within each group were further examined by
the chi-squared automatic interaction detection (CHAID)
method,58 using the likelihood ratio chi-square statistic as
growing criteria, along with a Bonferroni 0.05 adjustment
of probabilities. All analyses were carried out with the
Statistical Package for Social Sciences Version 17 software
(SPSS, Inc., an IBM Company, Chicago, Illinois).
Results
Clinical
In the control group, one of the 11 implants was ulcerated;
none had developed clinical CC. In the COS group, three of
the 11 implants were ulcerated, and no cases of CC were
observed. The chitosan group had one ulcerated implant,
and all 11 implants had developed Baker grade III/IV capsular contracture (Figure 1B). All chitosan group capsules were
extremely thick, opaque, stiff, and resistant to cutting (Figure
1C). They were constricted, and surface folding was observed.
Histology
The average capsular thickness was 0.418 ± 0.160 mm in
the control group, 0.6364 ± 0.216 mm in the COS group,
and 2.746 ± 0.817 mm in the chitosan group. Capsular
thicknesses were found to be statistically different among
the three groups: capsular thicknesses from the control
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544
Aesthetic Surgery Journal 31(5)
Table 1. Outcomes for Capsule Inflammation of Control Versus
Experimental Groups
Table 2. Bacteria Isolated From Capsule and Implant Samples Removed
From All Sacrificed Rabbits
Group
Type of Inflammatory
Cells
%
Intensity
Control
Mononuclear
9.1
Mild
72.7
27.3
Chitooligosaccharide
Chitosan
Number (%) of Positive Cultures
%
Polymorph
36.4
Moderate
Mixed
54.5
High
0.0
9.1
Mild
54.5
45.5
Mononuclear
Polymorph
27.3
Moderate
Mixed
63.6
High
0.0
0.0
Mild
36.4
63.6
Mononuclear
Polymorph
45.5
Moderate
Mixed
54.5
High
Bacteria
Coagulase-negative staphylococci
Staphylococcus aureus
Bacillus gram-negative
0.0
Enterococcus
group were different from both the COS group (p = .035)
and the chitosan group (p = .003); capsular thicknesses
were also different between the COS and chitosan groups
(p = .003). No significant differences were observed
regarding the type of inflammatory cells or the intensity of
capsule inflammation among the groups (Table 1).
Apoptotic cells and necrosis (Figure 1D) were observed
strongly in the chitosan group. Fibrosis was a component of
all capsules, and no significant difference was found regarding the organization of collagen fibers, fusiform cell density,
or angiogenesis among all groups. Regarding the characteristics of connective tissues (either loose or dense), significant
differences were found between the control and the chitosan
groups (p = .001). The control group had less than 25%
density of connective tissue, and the chitosan group had
more than 25% dense connective tissue.
Microbiology
Bacteria were isolated from 36.4% (12 of 33) of the capsules and from 78.8% (26 of 33) of the implants. The
organisms cultured (Table 2) included coagulase-negative
staphylococci, S. aureus, gram-negative bacilli, and
Enterococcus spp. Among capsules that yielded bacteria,
11 of 12 harbored coagulase-negative staphylococci
(91.7%); enterococci were associated with one capsule
(8.3%). The same trend was observed in excised implants.
In 20 of 26 implants that yielded bacteria, coagulase-negative staphylococci were cultured from 76.9%, and
Enterococcus spp. was associated with one capsule (3.8%).
In contrast to the capsules, four of 26 bacteria-contaminated
implants harbored gram-negative bacilli (15.4%), and one
of 26 demonstrated evidence of S. aureus (3.8%).
Overall, 39.4% (13 of 33) and 63.6% (21 of 33) of
culture-positive capsules and implants, respectively,
Capsules
Implants
Control
4 (36.4)
9 (81.8)
COS
5 (45.5)
8 (72.7)
Chitosan
2 (18.2)
3 (27.3)
Control
0 (0)
0 (0)
COS
0 (0)
1 (9.1)
Chitosan
0 (0)
0 (0)
Control
0 (0)
2 (18.2)
COS
0 (0)
1 (9.1)
Chitosan
0 (0)
1 (9.1)
Control
0 (0)
1 (9.1)
COS
1 (9.1)
0 (0)
Chitosan
0 (10)
0 (0)
COS, chitooligosaccharide.
yielded a single isolate; 0% (zero of 33) and 9.1% (three
of 33) yielded more than one. No fungi were recovered
from either capsules or implants.
No significant differences in the frequency of culture
positivity or the type of bacterial isolates were observed
among all study groups, nor was any significant association between microbial presence and histological data
observed.
With regard to skin isolates, the predominant isolate
was again coagulase-negative staphylococci, which were
formed in all rabbits. Bacterial isolates from skin were
similar to those from capsules and implants. Coagulasenegative staphylococci and gram-positive bacilli were
isolated from all operating room air samples, along with
Penicillium spp. and Aspergillus spp.
Immunology
Interstitial fluid of IL-8 levels decreased to the following:
89.4 ± 26.7 mg/mL in the control group, 78.3 ± 32.7 mg/
mL in the COS group, and 66.8 ± 17.9 mg/mL in the chitosan group. Significant differences were observed in IL-8
levels between the control and chitosan groups (p =
.028).
Levels of TNF-α decreased to the following: 143.9 ±
123.8 mg/mL in the control group, 96.8 ± 38.5 mg/mL in
the COS group, and 81.5 ± 31.8 mg/mL in the chitosan
group. Statistical analysis revealed no significant differences in the dialysate levels of TNF-α among all groups.
There was a correlation between IL-8 and TNF-α in the
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Marques et al
545
control group (p < .001) but not in the COS group (p =
.073) or the chitosan group (p = .099).
Discussion
In this study, we report on the development of CC in a
rabbit model associated with chitosan. All chitosan group
implants demonstrated clinical Baker grade III/IV breast
contractures with significantly thicker capsules than nontreated implants. Chitosan-exposed capsules were opaque,
stiff, and resistant to cutting, and considerable shrinkage
and folding of the implant surfaces were observed. These
characteristics may indicate the constricting nature of
fibrous implant capsules. Control (untreated) capsules
demonstrated thin capsule thicknesses, and the connective tissue had less than 25% dense tissue, compared to
the more than 25% dense connective tissue observed with
the LMWC-exposed capsules. This is consistent with the
fact that the major component of chitosan, glucosamine,
forms in cartilage tissue and is also present in tendons and
ligaments.59
The collagenous layer of granulation tissue is increased
with chitosan application; according to this finding, chitosan may stimulate fibroblast proliferation and extracellular matrix production.60 Chitosan has been shown to
induce an accelerated wound-healing process that did
increase TGF-β1, which had several proinflammatory regulatory influences such as cell migration, granulation tissue formation, and increased collagen production32 and
was a central mediator of fibrosis.61
A mixed/polymorph type of inflammatory cells was the
most common finding in all rabbit capsules, and inflammation was moderate/mild in all capsules. This finding
was expected, as chitosan is a chemoattractant for neutrophils.28,62 Chitosan enhanced the function of inflammatory cells such as polymorphonuclear leukocytes (PMN),
macrophages, fibroblasts (production of IL-8), angioendothelial cells,60 and had a systemic effect.63 Apoptotic
cells and necrosis were observed strongly in chitosan
implants, consistent with other reports.64,65
Statistical analyses revealed no significant differences
in the frequency of culture positivity and bacteria type
among the groups. Interestingly, no significant associations between microbial presence and histological data
were observed in any group. Similar bacterial isolates
were cultured from the rabbit skin and air samples, and
the predominant isolates were coagulase-negative staphylococci. The antimicrobial activity of chitosan and its
derivatives against several bacterial species has been recognized and considered one of the most important properties linked directly to their possible biological
applications25-28; however, recent studies investigating chitosan as a delivery method for drugs such as antibiotics66-69 questioned the high efficacy of chitosan alone as an
antibacterial agent. This study supports the idea that CC
formation is not the result of bacterial infection alone, in
contrast to the infectious hypothesis that has been championed and consistently supported by Burkhardt et al.1,3,70
To gain insight into the inflammatory process, major
biomarkers TNF-α and IL-8 were measured. This is the
first report examining extracellular levels of IL-8 and TNF-α
in a breast capsule implant environment. Microdialysate
levels of IL-8 were decreased (p < .05) in the chitosan
group as compared to the control group. No significant
differences in the microdialysate levels of TNF-α were
observed among the groups. In the control group, a correlation between IL-8 and TNF-α was observed; no significant
correlation between IL-8 and TNF-α levels was observed
in the experimental groups.
We originally hypothesized that serum concentrations
of the inflammatory mediators would be significantly
increased in the chitosan group due to the expected
greater inflammatory response with chitosan, as this molecule promotes the production of IL-8.60 The actual data
results did not support our hypothesis but were consistent
with a study from Tilg et al,71 who reported increased IL-8
and TNF-α levels in bacterial infection and decreased IL-8
and TNF-α levels in acute rejection. Interestingly, we
found clinical Baker grade III/IV breast capsule contractures in all rabbits exposed to chitosan associated with
acute (polymorph) and subacute (mixed) inflammation,
not due to a bacterial infection.
Not all chitosan implants were infected, and IL-8 and
TNF-α were decreased in the chitosan group. Molecular
regulation of IL-8 production has been studied in vitro,
and TNF-α has proven to be a major regulatory molecule.
It is not surprising that in vivo IL-8 and TNF-α serum levels were also significantly correlated in the control group.
The correlation between IL-8 and TNF-α has been well
established in the case of bacterial infection, less pronounced in cytomegalovirus hepatitis, and not apparent in
acute cellular liver rejection episodes. Lack of correlation
in acute rejection was also associated with low levels of
IL-8.71 This suggests that, in contrast to bacterial infection,
countering cytokines may be active in CC (at least promoted by chitosan), downregulating IL-8 transcription
and/or translation.
So far, no reports exist on the production and regulation
of IL-8 in CC. Recent studies have demonstrated that COS
displayed anti-inflammatory properties in immunocytes,
including the inhibition of nitric oxide, the downregulation of IL-6 and TNF-α, and the increase of cell viability of
neutrophils.72,73 Additionally, IL-8 was induced by a wide
range of stimuli, including lipopolysaccharide (LPS), a
component of the outer membrane of gram-negative bacteria and TNF-α. Lund et al74 concluded that LPS induced
IL-8 release in monocytes, whereas TNF-α was a good
inductor of IL-8 in PMN. In the chitosan contracture
model, we found decreased levels of IL-8, and it was possible to conclude that there was no gram-negative bacteria
infection to induce IL-8. On the other hand, chitosan
increased the production of TGF-β1,32 a central mediator
of fibrosis; the degree of CC is directly related to an
increased level of TGF-β.55 Even with contradictory studies
about the role of TNF-α, Morimoto et al75 concluded that
TNF-α played a pivotal role in the maintenance of hemostasis and tissue repair by inhibiting TGF-β1.
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546
Aesthetic Surgery Journal 31(5)
Our data support the theory that chitosan initiates CC
response due to a toxic local effect that results in an
impaired wound-healing response. An earlier series of
pilot studies were performed with much higher levels of
chitosan (data not shown). Using a similar experimental
protocol in the rabbit model, implants exposed to 25.0mg/mL levels were implanted. The majority of animals
expired within a short time period; surviving animals had
decreased weight (15%-25.8%) compared to baseline
body weights, with leukocytosis and decreased hemoglobin. At autopsy, fat biopsies were atrophied, and liver
specimens had lymphoid infiltration in the portal spaces.
We found toxicity with 25.0 mg/mL of implanted LMWC
per rabbit. The study design was modified to test decreased
chitosan levels that were not systemically toxic to the animals. In the reported data, all animals were clinically
healthy. Literature data reporting general toxicity testing
for chitosan are limited,31 and our results are consistent
with the few studies about chitosan toxicity.60,63,76-78
In several important studies,15,79 each rabbit received different implants. Darouiche et al,15 with the objective of
examining in vivo the antimicrobial efficacy of minocycline/rifampin–impregnated saline-filled silicone implants,
placed four implants in each rabbit (two antimicrobeimpregnated and two control implants). Shah et al,79 who
examined the infectious hypothesis in vivo, gave each rabbit a Staphylococcus epidermidis–contaminated implant and
a control implant. Despite the fact that this type of protocol
is well supported in the literature, due to the systemic influence of chitosan, the use of three different implants in the
same rabbit in our study obviously had the potential to
confound the results.
To clarify this issue, a control limb study was performed
(data not shown) and compared with the control group
from this study. Using a similar experimental protocol, 10
rabbits were implanted with two textured breast implants.
Interestingly, results from the control group in the main
study showed a lower capsular thickness than the control
limb group (0.81 ± 0.21 mm; p = .001). No significant differences were observed regarding the intensity of inflammation, characteristics of connective tissue (either loose or
dense), fusiform cell density, or angiogenesis between the
groups. However, significant differences were observed
with respect to the type of inflammatory cells, with a mixed
type of inflammatory cells found in 54.5% of the control
group in this study and mononuclear type of inflammatory
cells found in 55.6% of the control limb group (p = .017).
Significant differences were also observed in the organization of the collagen fibers, which were arrayed in sequence
in the control group of this study and haphazard in the
control limb group (p = .007). Statistical analysis revealed
no significant differences in the type or frequency of bacteria between the control group of this study and the control
limb group. Decreased levels of IL-8 (p = .016) and TNF-α
(p = .001) were observed in the control group of this study
when compared with the control limb group, which proves
the systemic influence of chitosan.
In a previous commentary,70 Burkhard considered that if a
rabbit model must be used for research, a more appropriate
model was the one reported by Shah et al,79,80 who used
bacterial contamination to produce contracture. In the Shah
et al study,79 16 New Zealand white rabbits each received a
S. epidermidis–contaminated implant and a control implant.
The capsules were dissected at two, four, six, and eight
weeks. Capsules on the contaminated side were two to three
times thicker than those on the control side, and they did not
change thickness with time. Capsules on the contaminated
side consisted of densely packed, longitudinally oriented
thick bundles of collagen fibers; there was a large cellular
infiltration with leukocytes and macrophages. By contrast,
the capsules on the control side were thinner and consisted
of loosely organized connective tissue fibers predominantly
parallel to the prosthesis surface. Bacteriological cultures on
the contaminated side consistently yielded S. epidermidis
with occasional diphtheroids, whereas the control side
showed no bacterial growth.
As reported in the Prantl et al81 study, we believe that
subclinical infection with chronic inflammation represents
one of the possible important reasons for the development
of CC. We also hypothesize that all possible causes of
fibrosis result in the common key factor of pathological
response with the development of chronic inflammation.
The Prantl et al81 study included only those implants with
high gel cohesiveness (third-generation implants); in these
implants, silicone filler presumably does not leak from the
shell into the tissue in the case of implant rupture.
Surprisingly, in 67% of their specimens, the authors
detected vacuolated macrophages with microcystic structures containing silicone. Also, in 54% of the specimens,
the capsular tissue contained empty spaces with varying
sizes of silicone particles. It remains unclear whether
these silicone structures represented friction particles from
the surface of the implant or particles from the implant
filler. Heppleston and Styles82 performed in vitro experiments demonstrating that silica damages macrophages,
which subsequently produce TGF-β1 and stimulate fibroblasts to produce collagen. However, since the Shah et al79
study, even with the many publications on infected
implants, we were unable to find any translation of the
Baker classification into a preclinical model.
An infection-induced contracture limb study was performed (data not shown) and compared with the chitosan
group of this study. Using a similar experimental protocol,
10 rabbits were implanted with two textured breast implants,
each one with a suspension of 100 µL of coagulase-negative
staphylococci (108 CFU/mL; 0.5 density on the McFarland
scale). Histologically, the average capsular thickness was
1.065 ± 0.287 mm in the infection-induced contracture
limb group (CoNS group) and 2.746 ± 0.817 mm in the
chitosan group. Capsular thicknesses were found to be statistically
different
among
the
two
groups
(p = .00003). A significant difference was also observed
regarding the type of inflammatory cells among the two
groups (p = .021), with the polymorph type being
predominant in the CoNS group and the mixed type being
predominant in the chitosan group. No significant differences were found between the two groups regarding the
intensity of capsule inflammation. Significant differences in
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Marques et al
547
angiogenesis were found between the CoNS and chitosan
groups (p = .004)—with absent/mild and moderate/ high
being equally present in the CoNS group but only high in
the chitosan group—as well as in the synovial metaplasia (p
= .043), which was always absent in the chitosan group
but present in some cases of the CoNS group. However, no
significant differences were found between the two groups
regarding the characteristics of the connective tissue (loose
or dense), the organization of the collagen fibers (parallel or
haphazard), or fusiform cell density (mild, moderate, or
high). Histologically, the type of CC induced by chitosan
was different from that induced by infection, in that (1) the
capsule was thicker, (2) the mixed type of inflammatory
cells was predominant, (3) angiogenesis was high, and (4)
the synovial metaplasia was absent. Our results contribute
a preclinical noninfectious model of CC to the literature, but
further studies are necessary.
We sacrificed the rabbits at four weeks to study early
capsule formation and to understand the possible models
of wound healing.39 A longer term study would be important and is planned. However, long-term differences in
capsule structures under these experimental challenges
result from different wound-healing trajectories from Day
0. Our strategy was to examine these early differences
with methods that were sensitive to detecting histological
or biomarker changes. There is no consensus about the
length of time necessary in a preclinical model. In a clinical mode, we proposed a follow-up period longer than 42
months.83 However, it might be expected that the finding
of a dense collagenous capsule would increase with time,
reflecting a continued stimulus toward a fibroplasia and
ultimate collagen remodeling.84-86
The weaknesses of this study include the relatively
small size and the lack of capsule immunohistochemistry
detection of IL-8 and TNF-α in tissue specimens.
Nevertheless, the release of IL-8 and TNF-α represented a
“spillage” of factors rather than a direct signal driving
inflammation and leukocyte recruitment; the use of microdialysis was appropriate for determining tissue concentration of cytokines such as IL-8 and TNF-α. Because of the
proximity of the sampling site to the source of the
cytokine, microdialysis provided a means of sensitively
detecting relative changes of inflammatory mediator concentration with experimental treatments.
Possible future studies would include a model in which
one silicone breast implant with ports (to measure the capsule pressure directly) impregnated with LMWC is implanted
per rabbit, as well as a model designed for detection of IL-8,
TNF-α, TGF-β1, and determination of a fibrosis index. We
previously reported complementary studies in which the
same protocol is used to analyze silicone breast implants
with ports impregnated with LMWC and those sprayed with
Tissucol/Tisseel (Baxter International, Deerfield, Illinois).87,88
Conclusions
Baker grade III/IV CC was observed in a rabbit model
when implants were impregnated with chitosan; the CC
was not due to a bacterial infection. This preclinical study
may provide a model to test various mechanistic hypotheses of breast capsule formation and subsequent CC and
suggests an approach of studying CC with a preclinical
animal model.
Acknowledgments
The authors thank Luis Sogalho, Pedro Lopes, Tom Powell,
Fernando Carvalho, Jiying Huang, Debby Noble, James Richardson, Anabela Silvestre, Pedro Leitão, Nuno Rego, Isabel
Santos, Cristina Moura, Elisabete Ricardo, Maria José Neto,
and Donna Henderson for their excellent assistance in organizing much of this work.
Disclosures
The authors declared no potential conflicts of interest with
respect to the authorship and publication of this article.
Funding
Research support was provided by the Faculty of Medicine,
Faculty of Sciences, Biotechnology Catholic at University at
Oporto and the Hospital of São João at Porto and Fundação
Ilídeo Pinho and Comissão de Fumento de Investigação em
Cuidados de Saúde Daniel Serrão at Portugal, as well as the
University of Texas Southwestern Medical Center at Dallas,
Texas, USA. Tissue expanders and implant devices were supplied by Allergan, Inc. (Santa Barbara, California) and Expo
Medica (Lisbon, Portugal).
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Publication
The Impact of Triamcinolone Acetonide
in Early Breast Capsule Formation in a
Rabbit Model
Marisa Marques, Spencer Brown, Inês
Correia-Sá, M. Natália D. S. Cordeiro,
Pedro Rodrigues-Pereira, Acácio
Gonçalves-Rodrigues, et al.
Aesthetic Plastic Surgery
ISSN 0364-216X
Aesth Plast Surg
DOI 10.1007/s00266-012-9888-z
1 23
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Aesth Plast Surg
DOI 10.1007/s00266-012-9888-z
The Impact of Triamcinolone Acetonide in Early Breast Capsule
Formation in a Rabbit Model
Marisa Marques • Spencer Brown • Inês Correia-Sá •
M. Natália D. S. Cordeiro • Pedro Rodrigues-Pereira •
Acácio Gonçalves-Rodrigues • José Amarante
Received: 19 November 2011 / Accepted: 27 February 2012
Ó Springer Science+Business Media, LLC and International Society of Aesthetic Plastic Surgery 2012
Abstract
Background The etiology and clinical treatment of capsular contracture remain unresolved as the causes may be
multifactorial. Triamcinolone acetonide applied in the
pocket during surgery was reported to be ineffective in
prevention of capsular contracture. However, if injected
4–6 weeks after surgery or as a treatment for capsular
contracture, decreased applanation tonometry measurements and pain were observed. It was assumed that intraoperative application of triamcinolone was not effective
because its effect does not last long enough. However,
betadine, antibiotics, and fibrin were found to be effective
in preventing capsular contracture with intraoperative
applications and are more effective in the early phases of
wound healing than in later stages. The role of triamcinolone acetonide in capsule formation is unknown. The
purpose of this study was to determine if triamcinolone
acetonide modulates breast capsule formation or capsular
contracture in the early phases of wound healing in a rabbit
model.
Methods Rabbits (n = 19) were implanted with one tissue expander and two breast implants and were killed at
4 weeks. Implant pocket groups were (1) Control (n = 10)
and (2) Triamcinolone (n = 9). Pressure/volume curves
and histological, immunological, and microbiological
evaluations were performed. Operating room air samples
and contact skin samples were collected for microbiological evaluation.
Results In the triamcinolone group, a decreased capsular
thickness, mild and mononuclear inflammation, and negative or mild angiogenesis were observed. There were no
significant differences in intracapsular pressure, fusiform
M. Marques I. Correia-Sá A. Gonçalves-Rodrigues J. Amarante
Faculty of Medicine, University of Oporto, Porto, Portugal
M. N. D. S. Cordeiro
Department of Chemistry, Faculty of Sciences,
University of Oporto, Porto, Portugal
e-mail: [email protected]
M. Marques (&) I. Correia-Sá A. Gonçalves-Rodrigues J. Amarante
Department of Plastic and Reconstructive Surgery, Hospital
of São João, piso 7, Alameda Prof. Hernâni Monteiro,
Porto, Portugal
e-mail: [email protected]
I. Correia-Sá
e-mail: [email protected]
J. Amarante
e-mail: [email protected]
P. Rodrigues-Pereira
Department of Pathology, Hospital of São João, Porto, Portugal
e-mail: [email protected]
A. Gonçalves-Rodrigues
Department of Microbiology, Faculty of Medicine,
University of Oporto, Porto, Portugal
e-mail: [email protected]
S. Brown
Department of Plastic Surgery Research, Nancy L. & Perry Bass
Advanced Wound Healing Laboratory, University
of Texas Southwestern Medical School, Dallas, TX, USA
e-mail: [email protected]
123
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Aesth Plast Surg
cell density, connective tissue, organization of collagen
fibers, and microbiological results between the groups.
There was no significant difference in the dialysate levels
of IL-8 and TNF-a, but correlation between IL-8 and TNFa was observed.
Conclusion Triamcinolone acetonide during breast
implantation influences early capsule formation and may
reduce capsular contracture.
Level of Evidence III This journal requires that authors
assign a level of evidence to each article. For a full
description of these Evidence-Based Medicine ratings,
please refer to the Table of Contents or the online
Instructions to Authors at www.springer.com/00266.
Keywords Breast capsule Triamcinolone acetonide Pressure Histology Microbiology Immunology
Capsule formation is a foreign body reaction that occurs in all
patients who have breast implants. Normally not thicker than
1-mm [44], the capsule is part of the normal healing process
and may help keeping the implant in place [21, 22]. Capsular
contracture (CC) remains the most severe complication with
silicone and saline breast implants, with an incidence ranging from 8 to 45 % [8, 23, 29, 33, 41]. The etiology of CC is
not completely understood, but it is thought to be multifactorial [4, 34]. Factors related to wound healing [49] and
infection [2, 3, 14, 18, 41, 47] are known to influence the
development of this clinical condition.
Etiology, prevention, and treatment measures for CC
have been extensively discussed, but there is no agreement
on a generally accepted therapeutic pathway. All the
reported clinical procedures used to minimize points of
contamination are crucial, and many plastic surgeons follow the general principles of the ‘‘Betadine Era’’ [2] and
the ‘‘Post-Betadine Era’’ [3, 5] to prevent CC. Betadine [2],
antibiotics [3, 5], and fibrin [35, 36] are clinically associated with a low incidence of CC and are more effective in
the early phases of wound healing. However, even when
following all the procedures proven to be effective for
diminishing this complication, it is still an important late
complication of breast implant surgery [41].
In preclinical studies, treatment with mesna [6], mitomicina C [24], zafirlukast [9, 46], pirfenidone [25], or
halofuginone [51] reduced capsule thickness, fibroblast cell
proliferation, and collagen deposition. Nevertheless, these
drugs are not commonly used in clinical practice, with the
exception of the zafirlukast. This drug is currently
approved for the treatment of asthma, but its role in the
treatment of CC is limited to severe cases due to the possibility of severe side effects [11, 28].
The capsule is known to be composed of a layer of
fibrous dense connective tissue [17] and is an integral part
123
of the wound-healing process. Although initially beneficial,
the healing process can become pathogenic if it continues
unchecked, leading to considerable tissue remodeling and
the formation of permanent scar tissue [13], as in CC.
Corticosteroids administered during wound healing have
been shown to stop the growth of granulation completely,
stop the proliferation of fibroblasts, diminish new outgrowths of endothelial buds from blood vessels, and stop
the maturation of the fibroblasts already present in connective tissue [7]. Also, when administered early after
injury, corticosteroids delay the appearance of inflammatory cells, fibroblasts, the deposition of ground substance,
collagen, regeneration of capillaries, contraction, and epithelial migration [20]. These data raised interest in the use
of steroids in the treatment and prevention of CC.
The data available in the literature regarding the role of
steroids in the prevention and treatment of CC is sparse and
contradictory. Perrin [38] reported less than 5 % of significant capsule formation in patients who underwent
augmentation mammaplasty with inflatable breast prostheses filled with saline and a cortisone derivative, with no
evidence of wound complications attributable to the steroid. These results were reinforced by those of Ksander
[31], who, in a preclinical model with rats, showed that
saline implants filled with saline solution were harder and
surrounded by a thicker capsular membrane than those
filled with methylprednisolone sodium succinate at 60 and
120 days.
On the other hand, Caffee et al. [16] reported in a preclinical study that putting triamcinolone in the pocket
during surgery was ineffective in the prevention of CC, but
if injected 4 and 8 weeks postoperatively (invasive
method), the drug was able to completely eliminate CC.
Caffee [15] also reported the effectiveness of postoperative
injection of triamcinolone in reducing the risk of recurrent
contracture in a high-risk group of patients. Sconfienza
et al. [43] demonstrated that US-guided injection of 40 mg
of triamcinolone acetonide (TA) into the peri-implant
pouch of women with augmented or reconstructed breasts
affected by Baker grade IV CC was effective in reducing
capsular thickness and the patient’s discomfort.
Although the data have been presented, the role of steroids in the treatment and prevention of CC is not completely understood. It is not clear whether steroids are
effective in preventing CC when placed in the implant
pocket, as the data available are inconsistent and contradictory. None of the clinical studies are prospective or
randomized. Moreover, none of the studies discussed here
established a clearly comprehensive role and mechanism of
steroids in the development of CC. Steroids have an
important role in the earlier phases of wound healing [20],
and the role of those effects on the early phase of breast
capsule formation are also not understood nor explored.
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Aesth Plast Surg
The main objective of this study was to perform a
comprehensive evaluation of the role of TA in capsule
formation in the early phases of wound healing [13] and the
histological, microbiological, and immunological characteristics in a rabbit model [4].
Materials and Methods
In an approved institutional animal care protocol, 19 New
Zealand white female rabbits were implanted with one
textured tissue expander (nonfilled; Allergan, Inc., Santa
Barbara, CA, USA) and two textured breast implants
(90 ml, Allergan). Prior to surgery, rabbit skin was washed
with BetadineÒ Surgical Scrub, which contains 7.5 %
povidone-iodine, followed by BetadineÒ solution, which
contains 10 % povidone-iodine (Purdue Pharma LP,
Stamford, CT, USA). The surgical procedure was performed in an animal operating theatre following aseptic
rules. Penicillin G 40,000 U/kg was administered intramuscularly intraoperatively. Talc-free gloves were used at
all times during the procedure. Two 5 cm incisions and one
2.5 cm incision were made directly over the skin and
subpanniculus carnosus to introduce the implants and the
expander, respectively. Pockets were developed in the
subpanniculus carnosus with atraumatic dissection along
the back region. Particular attention was paid to hemostasis
under direct vision, avoiding blunt instrumentation, and
there was no obvious bleeding. A sterile Op-site dressing
was placed over the skin around the incision before
inserting the tissue expander and the implant to avoid
contact with the skin. Wearing a new pair of talc-free
gloves, the surgeon introduced the implants and the tissue
expander with intact connecting tube and port. In the
experimental group, triamcinolone acetonide (Trigon
depotÒ, Bristol-Myers Squibb, New York, NY, USA) was
introduced into the implant and expander pocket. All
wounds were closed with two planes of interrupted suture.
The rabbits were divided into two groups: (1) the control
group with untreated implants and expander (n = 10), and
(2) the triamcinolone group which had the introduction of
1 ml (40 mg) of TA into each implant pocket and 0.25 ml
(10 mg) of TA into each expander pocket (n = 9). No fluid
suction was performed to retain the TA (TrigonÒ depot) in
the surgical pocket.
Rabbits were killed at 4 weeks. Before that, each animal
was anesthetized and the dorsal back area was shaved. A
pressure-measuring device (Stryker Instruments, Kalamazoo, MI, USA) was connected to the tissue expander port
and intracapsular pressures were recorded at each 5 ml
increment before any incision made to the capsule. Then, a
5-mm incision was made directly over the implant through
skin, panniculus carnosus, and capsule. A 100,000
molecular weight cutoff microdialysis probe (CMA
Microdialysis, Stockholm, Sweden) was placed near the
capsule–implant interface and microdialysates were collected using sterile normal saline solution (6 ll/min) for
1 h. Whole blood was obtained by venipuncture and serum
was collected after centrifugation (2,0009g min-1, 4 °C).
All capsule samples underwent histological and microbiological evaluation and all implants and expander devices
also underwent microbiological evaluation.
Microbiological Assessments
Air
Operating room air samples (n = 24) were collected during
all surgical procedures using the MAS 100-Eco air sampler
(EMD Chemicals, Inc., Gibbstown, NJ, USA) at a flow rate
of 100 l/min. Identification of bacterial and fungal isolates
followed standard microbiological procedures. Gram-positive cocci were characterized by biochemical methods.
Catalase-positive and coagulase-positive isolates were
reported as Staphylococcus aureus; catalase-positive and
coagulase-negative isolates were reported as coagulasenegative Staphylococci. Gram-negative bacilli were characterized with Vitek 2 software (VT2-R04.02, bioMerieux,
Inc., Durham, NC, USA). Fungi (molds) were characterized
according to macroscopic and microscopic morphology.
Rabbit Skin
A total of 57 contact plates were pressed to the shaved
dorsal skin surfaces (19 brain–heart agar, 19 mannitol salt
agar, and 19 Sabouraud agar contact plates). Brain–heart
and mannitol salt agar plates were incubated for 3 days at
28 °C, and Sabouraud plates were incubated for 7 days at
28 °C. The identification of the bacteria and fungi followed
the procedures reported above.
Capsules/Implants/Tissue Expanders
Excised tissue expanders/implants, and representative capsule samples were incubated at 37 °C for 3 days in brain–
heart broth and examined daily. Changes in the turbidity of
the broth media were considered positive and were subcultured in solid agar media. Characterization of microbial
isolates followed the above-described procedures.
Histological Assessment
Capsule specimens were fixed with 10 % buffered formalin
and embedded in paraffin. Sections were stained with
hematoxylin and eosin and evaluated histologically for
tissue inflammation and capsular thickness. Both the type
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of inflammatory infiltrate and the intensity were analyzed.
The inflammatory cells was grouped into three categories:
(1) mononuclear (lymphocytes, plasmocytes, and histiocytes), (2) mixed (mononuclear cells and eosinophils), and
(3) polymorph (eosinophils and heterophils/neutrophils).
Inflammatory infiltrate intensity was categorized according
to the following criteria: absent (-), mild (?), moderate
(??), and severe (???) [44].
Samples were stained with Masson’s trichrome to
characterize the connective tissue (loose or dense), the
organization of the collagen fibers (arranged in a parallel
array or haphazardly), angiogenesis (absent, mild, moderate, or high), and fusiform cell density (mild, moderate, or
high). The dense connective tissue was semiquantitatively
analyzed as (a) B25 % with thick collagen bundles less
than 25 %, (b) 25–50 %, (c) 50–75 %, and (d) [75 %.
Microdialysis Assessment
TNF-a levels were determined using Invitrogen’s Hu
TNF-a (catalog No. KHC3014:1; Life Technologies, Inc.,
Carlsbad, CA, USA). The assay was a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) in
which 100 ll of microdialysis fluid was pipetted into each
well. The protocol for IL-8 was performed using the BioSource Hu IL-8 US kit (catalog No. KHC0083/KHC0084;
Life Technologies).
Data Analysis
Data were analyzed by groups: Control (n = 20) and Triamcinolone (n = 18). One-way analysis of variance
(parametric or nonparametric) was performed to check
whether the several means of continuous variables (histologically measured thickness and dialysate levels of IL-8
and TNF-a) were equal, followed by post hoc range tests to
identify homogeneous subsets across groups. A two-tailed
independent paired t-test and the nonparametric alternative
Mann–Whitney U test were used to determine whether
such continuous variables were likely to show differences
between control and experimental groups. Categorical
variables were evaluated by v2 statistics and by /,
Cramer’s V, and contingency coefficients. Statistical significance was presumed at p B 0.05, and all analyses were
carried out with SPSS software (SPSS, Inc., Chicago, IL,
USA).
not shown). The expanders were included in the protocol to
determine the pressure–volume curves.
Clinical
In the triamcinolone group (Fig. 1), the capsules were thinner and more transparent than those of the control group.
Pressure
During pressure measurements, five (50 %) capsules ruptured in the control group. To avoid too little sampling, the
ruptured capsules were not excluded from statistical analyses; however, in such cases, the pressure value measured
before rupturing was maintained after further additional
saline was added. Pressure–volume curves were generated
for all rabbits that were killed. Statistical analyses revealed
no significant differences between the triamcinolone group
and the control group (Fig. 2).
Histology
Significant decreased capsular thickness was registered for
the triamcinolone group compared with the control group
(p B 0.001) (Table 1). A mixed cells were the most common finding in the control group and mononuclear cells
were the most common finding in the triamcinolone group
(Table 1). Significant differences were found between the
control group and the triamcinolone group (p = 0.0003).
A significant difference was observed between the triamcinolone and control groups (p = 0.009) with respect to the
Results
Statistical analyses revealed no significant differences in
the histological, immunological, and microbiological
results between breast implants and tissue expanders (data
123
Fig. 1 Capsule in the triamcinolone experimental group
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Aesth Plast Surg
between the control and triamcinolone groups with respect to
either implants or capsules. Also, there was no significant
association between microbial presence and histological data.
The predominant isolate was undoubtedly coagulase-negative
Staphylococci, which was identified predominantly in the
removed implants (Table 2). Isolated bacteria from the rabbits’
skin and from the operating room air were statistically similar
to those from the removed capsules and implants, with coagulase-negative Staphylococci prevailing.
No fungi were recovered from the removed capsules,
implants, or skin samples of all rabbits. Fungal species,
such as Penicillium spp. and Aspergillus, were recovered
from the operating room air.
Fig. 2 The pressure–volume curves
Immunology
intensity of inflammation, which was mild in the triamcinolone group and moderate in the control group (Table 1). No
significant differences in the fusiform cell density, connective tissue, and organization of the collagen fibers were
observed between the control and triamcinolone groups.
Significant differences were found in angiogenesis between
the control group, where it was basically moderate or high,
and the triamcinolone group (p = 0.007), where it was
negative or mild.
Microbiology
Statistical analysis revealed no significant difference in the type
of bacteria and in the frequency of culture positivity for bacteria
The dialysate levels of IL-8 decreased from 115.56 ±
128.03 mg/ml in the control group to 54.41 ± 31.21 mg/
ml in the triamcinolone group. Statistical analysis revealed
no significant difference in the dialysate levels of IL-8
between the control group and the triamcinolone group.
The dialysate levels of TNF-a decreased from 328.62 ±
307.55 mg/ml in the control group to 148.9177 ± 211.
92273 mg/ml in the triamcinolone group. Statistical analysis revealed no significant difference in the dialysate
levels of TNF-a between the control group and the triamcinolone group. There is a correlation between IL-8 and
TNF-a in the control group (p \ 0.001) and in the triamcinolone group (p = 0.036).
Table 1 Outcomes for capsular thickness and inflammation of control vs. triamcinolone groups
Group
Capsular thickness (mm)
Control
0.81 ± 0.209
Triamcinolone
0.53 ± 0.136
Type of inflammatory cells
(%)
Intensity
(%)
Mononuclear (chronic)
25.0
Mild
30.0
Polymorph (acute)
0
Moderate
70.0
Mixed (active chronic)
75.0
High
0
Mononuclear (chronic)
83.3
Mild
72.2
Polymorph (acute)
0
Moderate
27.8
Mixed (active chronic)
16.7
High
0
Table 2 Bacteria isolated from capsule and implant samples removed from all sacrificed rabbits
Bacteria
Coagulase-negative Staphylococci
Staphylococcus aureus
Bacillus gram-positive
Group
No. of positive cultures
Capsules (%)
Implants (%)
Control
2 (10)
13 (65)
Triamcinolone
6 (33)
14 (78)
Control
2 (10)
2 (10)
Triamcinolone
2 (11)
2 (11)
Control
Triamcinolone
1 (15)
2 (11)
1 (5)
2 (11)
Data collected from control (10 rabbits; 20 capsules and 20 implants) and triamcinolone (9 rabbits; 18 capsules and 18 implants) groups
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Discussion
The capsule that forms around the breast implant is composed by a layer of fibrous dense connective tissue [17],
and is an integral part of the wound-healing process. To
understand the formation of this late complication and the
potential therapeutic roles of both pharmacological and
nonpharmacological treatment approaches, it is crucial to
know the physiological mechanisms that are behind the
process that causes the formation of capsules.
Wound healing has been divided into three distinct
phases: inflammation, proliferation, and maturation [42].
The first phase of wound healing, which begins immediately upon injury through day 4–6, is characterized first by
hemostasis, an important event that serves as the initiating
step of the healing process; and an inflammatory response.
The second phase of wound healing (proliferative phase) is
characterized by epithelialization, angiogenesis, and provisional matrix formation and courses from day 4 through
14, overlapping phases 1 and 3. Fibroblasts and endothelial
cells are the predominant proliferating cells during this
phase. The maturation and remodeling (phase 3), which
occurs from day 8 through 1 year, is characterized by the
deposition of collagen in an organized and well-mannered
network [13].
As seen before, corticosteroids are known to have an
important role in wound healing, as they can stop the
growth of granulation completely, stop the proliferation of
fibroblasts, diminish the new outgrowths of endothelial
buds from blood vessels, and stop the maturation of the
fibroblasts already present in connective tissue [7]. Also,
when administered soon after injury, corticosteroids delay
the appearance of inflammatory cells and fibroblasts; the
deposition of ground substance, collagen, and regenerating
capillaries; contraction; and epithelial migration [20]. Steroids can have an important role in CC formation in both
the early and the late phase of fibrous phase formation.
The efficacy of triamcinolone in treating and preventing
CC in women has been reported [15, 43]. However, this
still represents an off-label practice and further studies are
required to validate the efficacy of this approach. Both
works have limitations: they were nonrandomized, with no
control group, had a limited follow-up period [15, 43], and
neither had as an objective the determination of the
mechanism of action of TA in capsular contracture formation. A comprehensive understanding of the effects of
TA on the mechanisms of capsular formation, the systemic
side effects, and the potential adverse events are, in our
opinion, crucial for the improvement of TA in clinical
activity.
This study is the first to analyze the impact of TA in early
capsule formation. We examined the effects of TA on pressure and histological, microbiological, and immunological
123
characteristics of capsules in an animal model to understand
the role of this steroid in early capsule formation and its
possible role in the prevention of CC. In our study, TA was
found to decrease capsular thickness upon macroscopic and
microscopic examination when compared to the control
group. These findings were also associated with decreased
inflammation and angiogenesis, as was expected, as steroids
are anti-inflammatory drugs capable of delaying the
appearance of inflammatory cells and they diminish the
proliferation of endothelium from blood vessels [7] and
regeneration of capillaries [20]. Although no significance
was found in the intracapsular pressure between the groups, a
tendency to lower pressures (and no capsule rupture during
the pressure measurement) was observed in the triamcinolone group compared to the control group (Fig. 2). Also, both
cytokine markers (IL-8 and TNF-a) were lower in the triamcinolone group, even without statistic significance. No
significant differences were observed in fusiform cell densities, connective tissue, or organization of collagen fibers.
Taken together, these results suggest that the introduction of
TA in the pocket intraoperatively has a role in capsule formation and might prevent CC.
Like Caffee et al. [16], we were not able to observe a
significant decreased capsular pressure in the group treated
with triamcinolone at the time of implant placement.
However, in our study we went further and analyzed not
only the pressure, an unquestionable indicator of capsular
contracture, but also other characteristics that are related to
the formation of this pathology as a continuous process.
The breast capsule begins to be formed after implant
placement; however, in clinical practice, the contracture is
a late complication, and follow-up, as long as 42 months
[33], is required to diagnose this entity. In preclinical
models, there is no consensus on the timing for sacrifice
and for representative stages for capsule formation. We
were not able to observe significant differences in pressure
between the groups, probably because we killed animals
too early. However, we were able to observe the early
alterations that are characteristic of capsule formation as
thinner and more transparent capsules on macroscopic and
microscopic evaluation and decreased inflammation and
angiogenesis. It might be expected and is reasonable to
assume that a more dense collagenous capsule with
increased thickness would be present with longer incubation times, reflecting a continued stimulus toward fibroplasia and ultimately collagen remodeling [10, 12, 45].
Caffee et al. reported in both preclinical [16] and clinical [15] studies that triamcinolone injected postoperatively
was able to eliminate CC and prevent the recurrence of this
condition. Those findings were confirmed by Sconfienza
et al. [43], who were able to demonstrate that US-guided
injection of triamcinolone acetonide in the peri-implant
pouch of women with augmented or reconstructed breasts
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Aesth Plast Surg
affected by Baker grade IV CC is effective in reducing
capsular contracture. Both authors concluded that triamcinolone was effective in the late stages of capsule formation. With our study we were able to observe that
triamcinolone is probably not only effective when injected
postoperatively, but also has a role in the early phases of
the development of capsular contracture.
In a previous report [36] that used the same protocol, the
authors were also able to find another compound, fibrin
(Tissucol/Tisseel) that was associated with a lower incidence of CC when sprayed in the pocket/implant during
surgery and was more effective in the early phases of
wound healing than in the later phases. It was found that
fibrin [36] was able to decrease intracapsular pressures
when compared to control (p B 0.001, data not shown),
and the capsular thickness was decreased (0.47 ± 0.129mm) (p B 0.001) as in triamcinolone group.
TNF-a plays an important role in the wound-healing
process. It is produced by activated macrophages, platelets,
keratinocytes, and other tissues and it stimulates mesenchymal, epithelial, and endothelial cell growth and endothelial cell chemotaxis [27, 32]. During the inflammatory
phase, it draws neutrophils into the injured area [39], generates NO [26] from macrophages, and digests damaged
extracellular matrix via matrix metalloproteinase [1]. During
the second phase, TNF-a upregulates KGF gene expression
in fibroblasts; upregulates integrins, a matrix component that
serves to anchor cells to the provisional matrix; stimulates
epithelial proliferation [32]; and is also a potent promoter of
angiogenesis. TNF-a is known to be a growth factor for
normal human fibroblasts and promotes the synthesis of
collagen and prostaglandin E2. IL-8 enhances neutrophil
adherence, chemotaxis, and granule release and enhances
epithelialization during wound healing [30, 32]. TNF-a
levels were reported to be markedly elevated in fibrotic
diseases such as liver fibrosis and is considered a mediator of
fibrosis like TGF-b1 [19]. Moritomo et al. [37] concluded
that TNF-a played a pivotal role in the maintenance of
hemostasis and tissue repair by inhibiting TGF-b1. We were
not able to find significant differences in IL-8 and TNF-a
levels, although decreased levels were observed in the group
treated with triamcinolone, possibly reflecting a role for this
drug in the modulation of the wound-healing process and
fibrotic response in the presence of the implant. More studies,
with longer follow-up and increasing doses of the compound,
are needed to confirm these data.
On the other hand, a significant correlation was also
found between IL-8 and TNF-a in both groups. This was
not unexpected, as correlations between IL-8 and TNF-a
with bacterial infections have been reported [48]. We did
not find any differences in the microbiology cultures
between groups, but further studies are necessary to clarify
whether triamcinolone increases the risk of infection.
With fibrin, a significant decrease in TNF-a
(140.9 ± 165.9 mg/ml) and IL-8 (23.9 ± 43.4 mg/ml)
levels (p = 0.003 and p = 0.048) was observed, supporting the possible role of this compound in early capsule
formation and in reduction of the collagen extracellular
matrix. No correlation between IL-8 and TNF-a was
observed in the fibrin group, which suggests a possible
antibacterial role of fibrin [36].
The main limitations of this study were (1) inappropriate
dosage in this model system; rabbits have much faster basal
metabolic rates than humans, and, as such, it is presumed
that rabbits have shorter drug half-lives [50]; (2) unknown
pharmacokinetics of triamcinolone in the capsule pocket
and subsequent metabolism, although triamcinolone modeling may be based on systemic steroid modeling [40]; (3)
short follow-up, as capsular contracture usually takes more
than 4 weeks to develop; and (4) the use of one tissue
expander per rabbit to directly measure the internal
expander pressures using the port. Silicone breast implants
with ports with a 90 ml volume capacity would be optimal
to achieve more accurate results but are not commercially
available. In addition, the preclinical model would not
support the use of multiple large expanders or implants
over long time periods. Our data do support future studies
examining triamcinolone as a potential agent for preventing CC.
Possible future studies may include (1) a preclinical
study using silicone breast implants with ports (to measure
the capsule pressure directly) and with introduction of
1.5 ml (60 mg) of triamcinolone acetonide into each
implant pocket and sacrifice of the animals at a much
longer time point with detection of IL-8, TNF-a, and
TGF-b1 and determination of fibrosis index; and (2) with
the same protocol, assessment of the effects of saline or
other vehicles of triamcinolone acetonide on pressure/volume curves. We believe that in a preclinical study, a higher
dose of triamcinolone acetonide introduced into the
implant pocket and a longer follow-up time period will
support the growing body of evidence that triamcinolone
acetonide mitigates capsular contracture.
In summary, our results suggest that triamcinolone has a
role in early capsule formation and it may have a role in the
prophylactic management of this complication. Obviously,
its role is centered on the management of the factors related
to wound healing [49], and it is important to exclude a
deleterious role in the factors related to infection that are
also known to increase CC [2, 3, 14, 18, 41, 47]. The
clinical intraoperative use of triamcinolone acetonide may
prove to be a reliable and safe way to prevent capsular
contracture in women undergoing breast implantation. The
ultimate goal is to translate these preclinical results to the
clinic, as these findings may help not only patients with
breast implants, but all patients with any device in which
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Aesth Plast Surg
capsule contracture around that device may lead to an
adverse clinical event.
Conflict of interest The authors have no conflicts of interest or
financial ties to disclose.
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123
Abstract Publication
Aesth Plast Surg
DOI 10.1007/s00266-012-9907-0
ABSTRACTS
Selected Abstracts from The Voice of Europe Session of the 4th
Annual Congress of the EASAPS
(Editorial Coordinator: Cristino Suárez López de Vergara)
Marketa Duskova • Salvatore Giordano • Asko Salmi • Delmar Henry •
Dirk F. Richter • Csaba Viczian • Huba Bajusz • Mario Pelle Ceravolo •
Georges J. Ghanimé • Marisa Marques • D. Jianu • M. Filipescu •
S. Adetu • Teresa Bernabeu • Selahattin Özmen • Cristino Suárez López de Vergara
Springer Science+Business Media, LLC and International Society of Aesthetic Plastic Surgery 2012
Metamorphosis
Marketa Duskova (Department of Plastic Surgery, Charles University,
Srobarova 50, 10034 Prague, Czech Republic,
email: [email protected])
The main aim of the aesthetic surgery is to improve quality of life. It
is known that less attractive people find it harder to obtain a good
personal and professional position in the society. The main point of
interest and the most important aspect is the face because human
attractiveness is specifically connected with facial appearance. In
considering correction of the facial visage with a great change, the
surgeon must pay attention, prepare meticulously with analysis of the
situation, and choose a suitable approach according to the circumstances as a whole. Then surgery must be performed with perfect
surgical technique, and the postoperative care must be carried out in
close cooperation with patient, perhaps also with other specialities or
even nonmedical experts.
The concrete process is shown in the case of a woman who
underwent complete profiloplasty (rhinoplasty, chin reduction), teeth
reconstruction, upper and lower blepharoplasty, augmentation of both
lips by synthetic implant, and application of injectable fillers into
facial wrinkles and rhytides. In addition, the beautician, hairdresser,
image consultant, and stylist put the last touches on the outcome.
Only such complex treatment may increase the patient’s mental
stability, self-confidence, and quality of life. The more perfect the
elimination of functional problems and stigmatizing disharmony, the
better are the preconditions for patients’ success and their assertion in
society.
M. Duskova S. Giordano A. Salmi D. Henry D. F. Richter C. Viczian H. Bajusz M. P. Ceravolo G. J. Ghanimé M. Marques D. Jianu M. Filipescu S. Adetu T. Bernabeu S. Özmen C. S. L. de Vergara (&)
Cirugı́a Plástica y Estética, Av. La Asunción, 30–28 izq.,
Santa Cruz, Tenerife, Spain
e-mail: [email protected]
Capsular Contracture After Cosmetic Breast Augmentation:
Do Topical Antibiotics Matter?
Salvatore Giordano (Department of Plastic Surgery, Turku University
Hospital, Turku, Finland), Asko Salmi (Department of Plastic
Surgery, KL Hospital, Helsinki, Finland)
Introduction: Antibacterial lavage with topical antibiotics may reduce
the occurrence of capsular contracture in breast implant surgery. A
retrospective analysis was performed to investigate this effect.
Materials and Methods: The study participants included 308 women
who underwent cosmetic breast augmentation during two different
periods: 2004–2008 (n = 168, group A) and 2009–2010 (n = 140,
group B). The same surgeon performed the surgery for all the women
using the inframammary approach and the dual-plane pocket. All the
patients had McGhan/Allergan 410 form stable textured implants. The
group A patients received antibiotics as a single perioperative intravenous dose of cephalothin 1.5 g and cephalexin 750 mg as an oral
course twice a day for 1 week after discharge. In the group B, perioperatively, 750 mg of cefuroxime was administrated intravenously.
Implants and pockets were irrigated with 10 ml of 10 % povidone–
iodine solution mixed with 750 mg of cefuroxime and 40 mg of
gentamicin. After discharge, 500 mg of levofloxacin was administered as an oral course once a day for 10 days. The postoperative
complications included occurrence of infection, seroma, and capsular
contracture. We considered capsular contracture significant when it
was graded Baker 3 or 4.
Results: The average postoperative follow-up period was 11 ± 13
months for group A and 3 ± 8 months for group B. No postoperative
infections or seroma were detected. Group B had no capsular contraction cases. The capsular contraction rate was significantly higher
in group A (5.9 vs. 0 %; p = 0.003).
Conclusions: The use of topical antibiotics in cosmetic breast surgery is
recommended because a significant increase in capsular contracture
was observed in patients not treated with topical antibiotics.
The Middle Third of the Face: Analysis, Techniques,
and Indications
Henry Delmar (90 Boulevard Du Cap, 06160 Cap D’Antibes, France,
email: [email protected])
The Aging Process: The aging process of the face acts in many modes
including squeletization, ptosis, and desequilibrium of muscular
123
Aesth Plast Surg
balance, with loosened tissues and lack of firmness and structure. This
gives modelization of the aging process in three modes: squeletization, ptosis, and fattening. This modelization gives the surgeon the
opportunity to propose an adequate association of techniques.
Malar Elevation: The indication of the ptosis mode is elevation of the
malar region. In 1994, we described, with F. Trepsat, a technique of
low malar suspension with the buccal approach, which allows correction of the ptosis and transfer of volume from low to high. The
aging process of the cheekbone is more superficial than deep. To
address this, many authors improve the technique with a suspension
of the orbicularis oculi by the palpebral approach. The goal for
traction of the orbicularis is a superficial lifting of the skin of the
cheekbone. But the weak point is a high traction in the palpebral
region, which results in a deformation of the glance, with palpebral
deformity. To enable correction for the superficial modification of the
cheekbone without palpebral deformity, we propose a new technique
with medical devices as follows:
•
•
•
Subperiosteal dissection of the cheekbone using the buccal
approach
Installation of medical devices both superficially and deep
Palpebral surgery and temporal lifting adapted to the indication.
This technique is called malar isolated positioning (MIP).
Indications: The indications for suspension of the cheekbone depend
on the aging lower eyelid and its treatment. Without treatment of the
lower eyelid, a buccal technique is recommended. In the situation of a
blepharoplasty, an eyelid approach and bone fixation are proposed.
The indications relate to the highness of the cheekbone. Indications
and results are shown.
What Can Be Achieved Through an Upper-Lid Incision?
Dirk F. Richter (Bonner Straße 84, Dreifaltigkeits-Krankenhaus,
50389 Wesseling, Germany, email: [email protected])
Upper-lid blepharoplasty, one of the most demanded aesthetic procedures, is not just treatment for dermatochalasis. The upper blepharoplasty incision can be used to adjust retro-orbicularis oculi fat and for
glabellar myotomy, lateral cantopexy, and browpexy (i.e., brow-lift).
The transblepharoplasty brow-lift is suitable for the lateral two thirds of
the brow. This technique is less invasive and allows an anchoring of the
underlying brow soft tissue to the bone. This permits stabilization or
elevation of the eyebrow without an endoscope because the nerves are
under direct vision.
Another approach is corrugator supercilii muscle resection through
a blepharoplasty incision, which is suitable for patients who have
significant corrugator hyperactivity and deep frown lines without
eyebrow or forehead ptosis. This procedure can be performed with or
without a concomitant blepharoplasty.
Through an upper-lid incision, the blepharoplasty as well as the
cantopexy, brow-lift, and resection of the corugator muscle can be
performed with a less invasive technique and fewer scars, which leads
to a high patient acceptance rate and satisfaction.
sagging but also from atrophy of soft tissues, particularly the orbital
septum and orbital fat. The evolution of orbital fat preservation and
the midfacial volumetric concept taught clinicians to treat the lower
eyelid with the midface as one aesthetic unit.
Methods: Besides the popular methods (arcus marginalis release, fat
medialization, septorhaphy, fat transfer), the authors present their
results with additional lateral tightening of the orbicularis oculi
muscle.
Discussion and Conclusion: To recreate a youthful appearance of the
lower lid, a clear indication for the choice of the correct operating
method is needed. Before the procedure, the anatomy around the
orbit, the eyelid laxity, the fat pads, the lid–cheek junction, and the
position of the midface must be analyzed. The lower lid vectors will
show the relationship between the anterior projection of the globe, the
lower lid, and the malar bony eminence. The authors give special
interest to the moderate elevation effect on the midface created by
additional lateral tightening of the orbicularis oculi muscle. The
authors present their algorithm, which may help in selecting the
correct procedures for the lower eyelid and midface operations.
Animation Deformities by Pectoralis Muscle: The Cinderella
of Submuscular Mammaplasty
Mario Pelle Ceravolo (Via Giovanni Severano 35, 00161 Rome, Italy,
email: [email protected])
Animation deformities are present in almost every patient submitted
to subpectoral augmentation mammaplasty. These deformities represent the most common complication related to the reported
operation and yet are the least known.
Animation deformities have been studied by the author in more
than 1,000 patients and classified according to clinical criteria in six
different categories. Many patients treated with the dual-plane technique present with animation deformity despite the ability of this
technique to avoid its occurrence.
The physiopathology of the deformity is related to the pulling
action of the muscle on the breast mass and not to implant dislocation
during the muscle contraction. The author presents his algorithm of
different techniques used for submuscular augmentation mammaplasty based on different anatomic preoperative situations.
Preservation of pectoralis muscle costal insertions, medial pectoralis nerve section for muscle denervation, and horizontal muscle
splitting are the main maneuvers used to avoid breast dynamic distortion. Horizontal muscle splitting consists of a horizontal incision
performed in the pectoralis muscle that splits it in two flaps. The
upper flap provides good coverage for the implant, whereas the lower
flap may be left attached to the chest to improve the projection of the
breast lower pole, or it may be rotated laterally or medially depending
on the clinical demand.
Horizontal muscle splitting is a personal technique that the author
has used during the last 10 years in more than 350 cases with aesthetically good results and a substantial decrease in the occurrence of
animation problems.
Conservative Rhinoplasty
The Challenging Lower Eyelid Correction: The Aesthetic Effect
of Lateral Orbicular Muscle Tightening
Csaba Viczian and Huba Bajusz (St. Gellert Private Clinic, 6722
Szeged, Kalvaria sgt 14, Hungary, email: [email protected])
Introduction: Aesthetic correction of the lower eyelids often is more
difficult and challenging than correction of the upper eyelids. The
characteristics of facial aging result not only from elastosis and
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Georges J. Ghanimé (Division of Plastic and Reconstructive Surgery,
Lebanese University, Faculty of Medicine, Lebanese Hospital,
Getawi, Beirut, Lebanon)
Currently, rhinoplasty is one of the most popular aesthetic surgical
procedures. This has led to refinement of the techniques, making them
simpler and more reliable and minimizing soft tissue trauma by using
the least invasive technique to accomplish the predetermined goals.
Aesth Plast Surg
Our experience includes more than 4,000 rhinoplasties performed
since 1992. The majority of the cases are managed by same-day
surgeries performed with the patient under general anesthesia using
only the closed approach. Because form and function work together, a
septoplasty is performed when there is septal deviation.
Our experience has led us to the conclusion that conservative
rhinoplasty is indicated in most cases.
Effects of Fibrin (Tisseel/Tissucol) on Breast Capsule Formation
in a Rabbit Model
Marisa Marques (Hospital de Sao Joao, Serviço de Cirurgia Plastica
(piso 7), Alameda Prof. Hernâni Monteiro. 4202 Porto, Portugal,
email: [email protected])
Background: The etiology and clinical treatment of capsular contracture remain unresolved because causes may be multifactorial. The
previously described environmental challenges that accelerate capsule
contracture have been bacteria, especially coagulase-negative staphylococci. The role of fibrin in capsule formation was controversial in
various independent studies. Study 1 aimed to influence capsule
wound healing with blood, fibrin, and thrombin, and to make a
comparison with a control group in a rabbit model implanted with
tissue expanders. To clarify the results of this first study, study 2 was
performed to determine whether fibrin and coagulase-negative
staphylococci modulated capsule formation in a rabbit model
implanted with a tissue expander and breast implants.
Methods: Study 1: Each New Zealand white rabbit (n = 18) received
four different tissue expanders and then was killed at 2 or 4 weeks.
The four study groups were the control, fibrin, thrombin, and blood
cohorts. Study 2: Rabbits (n = 31) were implanted with one tissue
expander and two breast implants and then were killed at 4 weeks.
The implant pocket groups included the control (n = 20), fibrin
(n = 22), and coagulase-negative staphylococci (CoNS) cohorts
(n = 20). Pressure and volume curves as well as histologic and
microbiologic evaluations were performed. Operating room air samples and contact skin samples were collected for microbiologic
evaluation.
Results: Study 1: At 4 weeks, significantly lower intracapsular
pressures were measured in the experimental fibrin and thrombin
groups than in the control group. For the control and fibrin groups,
mixed inflammation was correlated with decreased intracapsular
pressures, whereas mononuclear inflammation was correlated with
increased intracapsular pressure. The predominant isolates in capsules, tissue expanders, and rabbit skin were coagulase-negative
staphylococci. For the fibrin and thrombin groups, cultures other than
staphylococci and negative cultures were correlated with decreased
intracapsular pressures, whereas staphylococci isolation was correlated with increased intracapsular pressures. Study 2: In the fibrin
group, significantly decreased intracapsular pressures, thinner capsules, loose or dense (\25 %) connective tissue, and negative or mild
angiogenesis were observed. In the CoNS group, increased capsular
thicknesses and a polymorph type of inflammatory cells were the
most common findings. Similar bacteria in capsules, implants, and
skin were cultured from all the study groups. A Baker grade 4
contracture was observed in an implant infected with Micrococcuss
spp.
Conclusion: Fibrin (Tisseel/Tissucol) was associated with reduction
of capsule formation in our preclinical animal model, which makes
fibrin an attractive potential therapeutic agent for women undergoing
breast implants. Clinical strategies for preventing bacterial contamination during surgery are crucial because low pathogenic agents may
promote capsular contracture.
Face and Neck Rejuvenation Using Combined Techniques: Laser
Lipolysis, Fractional Laser, Liposuction, and Lipofilling
Dana Jianu, M. Filipescu, S. Adetu (ProEstetica Medical Center, 38–
40. Tudor Stefan Street, Bucarest, Romania, email:
[email protected])
Background: This study assessed the role for the combined use of
fractional laser (CO2 laser) and laser lipolysis (980-nm diode laser)
for face and neck rejuvenation.
Methods: From September 2008 to February 2011, 39 subjects
underwent laser treatments for facial and neck rejuvenation. The
treatment consisted of using laser lipolysis (980-nm diode laser MedArt), sometimes with additional facial fractional laser CO2 MedArt.
Laser lipolysis was performed to restore the jaw line and the mandible–neck angle respectively for laxity of the jaws and the anterior
cervical part. After tumescent anesthesia, a 1.5-mm-diameter needle
(80 mm long) housing a 600-lm optical fiber was inserted into the
subcutaneous fat. The cannula was moved in predetermined lines to
obtain a homogeneous distribution in the treated area. The laser settings were 10–11 W in relation to the thickness of the subcutaneous
fat and dermis. In some cases, additional fine liposuction and lipofilling were necessary. The settings for the fractional laser used in
face rejuvenation usually provided a 10-W, medium-density beam for
4 ms. For eyelids, the settings provided an 8-W, high-density beam
for 5 ms.
Results: A total of 108 laser lipolysis procedures were performed for
39 patients. The areas treated were the jaws (9 patients) and the jaws
together with the anterior part of the neck (30 patients). The mean
cumulative energy was 1,800 J for the jaw area and 3,000 J for the
neck. Contour correction and skin retraction were noted after
4–7 days for almost all the patients.
Conclusion: This clinical study demonstrates that removal of fat in
small volumes with concurrent subdermal tissue contraction can be
performed safely and effectively using a 980-nm diode laser. Additional benefits include excellent patient tolerance and a quick
recovery time. The study also confirms that accumulated energy
derived from fractional laser combined with laser lipolysis is safe and
can improve the contraction and skin regeneration, leading to a better
rejuvenation of the face and neck.
Combined Mastopexy and Breast Augmentation
Teresa Bernabeu (avda. Benidorm 19, Ed. Arena piso 8, 03540,
Alicante, Spain, email: [email protected])
Background: Combined mastopexy and breast augmentation, first
described by Gonzalez Ulloa [1] and Regnaul [2] in 1960, has seen an
increase in demand in recent years. Whereas a woman previously was
satisfied with a mastopexy alone, currently, the patient herself
demands the combination of filling and lifting of the breast in a single
procedure with the smallest possible scar. These patients are, without
doubt, influenced by the increasingly widespread images of the aesthetic appearance conferred by breast implants and the growing trend
to maintain C- or D-cup breasts. Driven by these demands, plastic
surgeons have increased the indications for this type of intervention
and the frequency of their use. These interventions present difficulties
and potential risks and can become absolute disasters [3]. The steps to
follow are selection of the patient, selection of the mastopexy technique, glandular resection, and implant selection, with all these steps
aimed at achieving the aesthetic objectives while leaving minimal and
inconspicuous scars.
Methods: The literature contains different rules [4–7] regarding the
selection of mastopexy technique based primarily on the distance
from the sternal notch and nipple to the areola and inframammary
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Aesth Plast Surg
fold together with the degree of breast ptosis. These rules are helpful,
although they may vary from one procedure to another and become
modified over time according to this author’s experience. In addition
to this, before surgery, there is a degree of uncertainty regarding the
choice of technique, which in many cases does not become clear until
the breast implant is in place. The method used to achieve a longerlasting result of mastopexy combines three factors: an anatomic
implant with its different projections and heights to help prevent
recurrence of ptosis, glandular resection as required, and the subfascial placement of the prosthesis, which produces greater concordance
and harmony between the implant and the mammary gland.
Results: The results obtained in the last 2 years with the aforementioned method are aesthetically better and have lower rates of
complications than those previously obtained by the author.
Conclusion: Subfascial positioning of anatomic implants with maximum projection and glandular resection as required help to provide
greater durability in mastopexy.
References
1. Gonzalez Ulloa M (1960) Correction of hypertrophy of the breast
by exogenous material. Plast Reconstr Surg 25:15.
2. Regnault P (1966) The hypoplastic and ptotic breast: A combined
operation with prosthetic augmentation. Plast Reconstr Surg
37:31.
3. Stevens WG (2007) One-stage mastopexy with breast augmentation: A review of 321 patients. Plast Renconstr Surg 120:1674–
1679.
4. Spear SL (2001) Concentric mastopexy revisited. Plast Reconst
Surg 107(5):1294–1299.
5. Cardenas-Camarena L (2006) Augmentation/mastopexy: How to
select and perform the proper technique. Aesthetic Plast Surg
30:21–33.
6. de la Fuente A (1992) Periareolar Mastopexy with Mammary
implants. Aesthetic Plast Surg 16: 337–341.
7. Spear SL. (1990) Guidelines in concentric mastopexy. Plast
Reconstr Surg 85:961.
Of Form, Function, and Aesthetics in Nose Surgery
Selahattin Özmen (Department of Plastic, Reconstructive,
and Aesthetic Surgery and Hand Surgery, Faculty of Medicine, Gazi
University, Ankara, Turkey)
Traditional rhinoplasty operations depend on cartilage, bone, or both,
and sometimes soft tissue resections. In modern nose surgery, however, the function and the aesthetic appearance should be seized
together.
Resections could be limited mainly to three cartilaginous areas,
with cartilages reconstructed in these areas: lower lateral (alar) cartilages, upper lateral cartilages, and septal cartilage.
Nasal Tip Region: Providing a natural-appearing nasal tip contour has
always been a key component of a successful rhinoplasty. One prerequisite for a successful rhinoplasty is nasal tip support and its
influence on nasal tip projection. Alar cartilages are the chief providers of structural support to the tip of both the nose and the external
nasal valve.
To reshape the nasal tip, we use the sliding alar cartilage (SAC)
flap, a novel technique for nasal tip contouring and support. The SAC
technique [1]
•
•
Provides an aesthetically acceptable and naturally good-looking
nasal tip and alar contour
Supplies effective nasal tip support and could be used for ‘‘pinch
nose’’ deformity
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•
•
•
•
•
•
Does not require any cartilage graft and thus results in no donorsite morbidity
Involves minimal or no risk for malposition, distortion, or
resorption because it is a flap secured with sutures
Results in a nonpalpable cartilage graft, in contrast to other
cartilage grafts, because it is prepared from the original alar
cartilage and placed under the caudal alar cartilage
Produces no unwanted effect on the external nasal valve function
because the connection between the upper lateral cartilages and
the alar cartilages is not broken
Reserves the cranial parts of the alar cartilages, allowing for their
use in the future whenever there is a need (e.g., septal
perforations)
Uses a flap with a double-layered alar cartilage, which can supply
more resistance against the thick tip in some noses that poses a
real challenge.
Middle Vault: Another point is the resection of the upper lateral
cartilages. Upper lateral cartilages are attached to the septum in an
obtuse angle forming a T shape. Dorsal hump reduction during rhinoplasty almost always breaks this connection and can create both
functional and aesthetic problems if performed incorrectly. We preserve the upper lateral cartilages using the upper lateral cartilage foldin flap technique [2]. This technique has a combined spreader or splay
graft effect without cartilage grafts.
The upper lateral cartilage fold-in flap technique might be applicable for almost all primary rhinoplasty patients because the previous
physiologic structure is reconstructed. It also is suitable for patients
who have not undergone previous dorsal hump removal.
To have a splay effect, only mucoperichondrial sutures should be
used, and at least a 1–2-mm middle nasal vault reduction is necessary.
For narrow noses, to prevent a very wide appearance in the middle
nasal vault, transcartilaginous mattress sutures should be used.
Suturing the mucoperichondrium over the cartilages could supply a
smoother dorsum at the middle vault.
Although it is possible to use this technique with closed rhinoplasty approaches, it is easier with the open approach. This technique
is not suitable for secondary rhinoplasty cases, in which upper lateral
cartilage resection has already been performed. In these cases,
spreader or splay grafts might be applied.
Nasal Septum: Septoplasty: Excessive resection of the septal cartilage
or bone leaving an L-strut is the technique most surgeons prefer. But
the weakened septum could collapse in a relatively minor trauma.
In most septal deviations, the problem is mostly related to the
bony septum, including the maxillary crest and the perpendicular
plate of the ethmoid bone or vomer. The cartilage usually is not
broken, only bent in an anteroposterior or craniocaudal direction. In
most cases, just releasing these bonding factors by removing deviated
bones and bone spurs leads to a relaxation in the cartilage, with
cartilaginous resection unnecessary or minimal.
On the other hand, the cartilage should be excised or reconstructed
if there is a fracture or cartilage excess.
Consequently, septal deviations should be corrected very meticulously, and septal cartilages and bones should not be excised when it
is not necessary. They should be reconstructed in-site and in an
extracorporeal fashion whenever needed.
References
1. Ozmen S, Eryilmaz T, Sencan A, Cukurluoglu O, Uygur S,
Ayhan S, Atabay K (2009) Sliding alar cartilage (SAC) flap: A
new technique for nasal tip surgery. Ann Plast Surg 63:480–485.
2. Ozmen S, Ayhan S, Findikcioglu K, Kandal S, Atabay K (2008)
Upper lateral cartilage fold-in flap: A combined spreader and/or
splay graft effect without cartilage grafts. Ann Plast Surg
61:527–532.